Anti-ilt4 antibodies and antigen-binding fragments

ABSTRACT

The present invention provides antibodies and antigen-binding fragments thereof that bind to ILT4 (immunoglobulin-like transcript 4) and combinations thereof, e.g., with an anti-PD1 antibody. Also provided are methods of use thereof, for example, for treating or preventing cancer in a subject; and methods of making such antibodies and fragments.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/483,019, filed on Apr. 7, 2017, the disclosure ofwhich is incorporated herein by its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies and antigen-bindingfragments thereof that bind to immunoglobulin-like transcript 4 (ILT4)as well as methods of making and using such antibodies andantigen-binding fragments, for example, to treat diseases such ascancer.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name 24443USDIV1-SEQLIST-03JUN2021.txt, creation date of Jun. 3,2021 and a size of 125 kb. This sequence listing submitted via EFS-Webis part of the specification and is herein incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

A common strategy used by tumor cells to escape innate and adaptiveimmune response is associated with aberrant expression of humanleukocyte antigen (HLA)-G (Curigliano et al. Clin Cancer Res. 2013 andGonzalez et al. Crit Rev Clin Lab Sci. 2012). HLA-G can directly inhibitimmune cell function through receptor binding and/or throughtrogocytosis and impairment of chemotaxis (Morandi et al. CytokineGrowth Factor Review. 2014 and Lin et al. Mol Med. 2015). Its highexpression in multiple tumor types, including for example, colorectal,pancreatic, endometrial, lung, breast, ovarian, and gastric cancer, isassociated with advanced disease stage, tumor invasiveness, metastaticpotential and an unfavorable prognosis (Lin et al. Mol Med. 2015. andLoumange et al. Int J Cancer. 2014). Antibody-mediated blockade of HLA-Gfunction in transgenic mouse models has been shown to inhibit tumordevelopment and block expansion of myeloid-derived suppressor cells(MDSC) (Loumange et al. Int J Cancer. 2014, Lin et al. Hum Immunol.2013, and Agaugue et al. Blood. 2011). HLA-G binding to ILT4 candirectly inhibit the function of monocytes, dendritic cells, andneutrophils, thus impairing the innate immune anti-tumor response. Theinteraction between HLA-G and monocytes due to ILT4 inhibits maturationof human monocyte-derived antigen-presenting cells (APCs) resulting in areduced expression of MHC class II antigens and co-stimulatory moleculesthrough Stat3 activation (Colonna et al. J Immunol. 1998; Allan et al. JExp Med. 1999, and Liang et al. Proc Natl Sci USA. 2008). Using humanmonocyte-derived dendritic cells (DCs) and ILT4-transgenic mice, HLA-Gwas shown to induce the development of tolerogenic APCs with arrestmaturation/activation of myeloid DCs, and the induction of tolerogenicDCs by HLA-G was through disrupting the MHC class II presentationpathway (Ristich et al. Eur J Immunol. 2005).

An unmet medical need exists for patients that do not respond to T-celltherapy but may benefit from relief of tissueassociated-macrophage/MDSC-mediated tumor tolerance (e.g. myeloid “rich”tumors). ILT4 blockade would fill this need and would differentiate fromcurrent T-cell-targeted antibodies (e.g. anti-PD1, anti-TIGIT) byrelieving suppression of tolerogenic myeloid cells in the tumormicroenvironment.

SUMMARY OF THE INVENTION

The present invention provides antibodies or antigen-binding fragmentsthereof that bind to human ILT4. In certain embodiments, the antibody orantigen-binding fragment thereof binds to one or more amino acidresidues in a human ILT4 epitope selected from the group consisting ofLYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID NO:61),and HTGRYGCQ (SEQ ID NO:62). In certain embodiments, the antibody orantigen-binding fragment thereof protects the epitope from deuteriumexchange with a deuterium source, such as D₂O. In one embodiment, theantibody or antigen-binding fragment thereof binds to one or more aminoacid residues in the epitope LYREKKSASW (SEQ ID NO:59). In anotherembodiment, the antibody or antigen-binding fragment thereof binds toone or more amino acid residues in the epitope TRIRPEL (SEQ ID NO:60).In yet another embodiment, the antibody or antigen-binding fragmentthereof binds to one or more amino acid residues in the epitope NGQF(SEQ ID NO:61). In still another embodiment, the antibody orantigen-binding fragment thereof binds to one or more amino acidresidues in the epitope HTGRYGCQ (SEQ ID NO:62). In yet still anotherembodiment, the antibody or antigen-binding fragment thereof binds toone or more amino acid residues in two, three, or four ILT4 epitopesselected from the group consisting of LYREKKSASW (SEQ ID NO:59), TRIRPEL(SEQ ID NO:60), NGQF (SEQ ID NO:61), and HTGRYGCQ (SEQ ID NO:62). In oneembodiment, the antibody or antigen-binding fragment thereof binds toone or more amino acid residues in the epitope LYREKKSASW (SEQ ID NO:59)and protects the epitope from deuterium exchange with a deuterium sourcesuch as D₂O. In another embodiment, the antibody or antigen-bindingfragment thereof binds to one or more amino acid residues in the epitopeTRIRPEL (SEQ ID NO:60) and protects the epitope from deuterium exchangewith a deuterium source such as D₂O. In yet another embodiment, theantibody or antigen-binding fragment thereof binds to one or more aminoacid residues in the epitope NGQF (SEQ ID NO:61) and protects theepitope from deuterium exchange with a deuterium source such as D₂O. Instill another embodiment, the antibody or antigen-binding fragmentthereof binds to one or more amino acid residues in the epitope HTGRYGCQ(SEQ ID NO:62) and protects the epitope from deuterium exchange with adeuterium source such as D₂O. In yet still another embodiment, theantibody or antigen-binding fragment thereof binds to one or more aminoacid residues in two, three, or four ILT4 epitopes selected from thegroup consisting of LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60),NGQF (SEQ ID NO:61), and HTGRYGCQ (SEQ ID NO:62) and protects theepitopes from deuterium exchange with a deuterium source such as D₂O.

The present invention also provides an antibody or antigen-bindingfragment thereof that binds to the same epitope of human ILT4 as anyantibody or antigen-binding fragment thereof disclosed herein. Incertain embodiments, the antibody or antigen-binding fragment thereofbinds to the same epitope of human ILT4 as an antibody orantigen-binding fragment thereof comprising the heavy chain and lightchain amino acid sequences set forth in SEQ ID NOs:1 and 3; 2 and 4; 2and 5; 2 and 6; 2 and 7; 2 and 3; 8 and 11; 9 and 11; 10 and 11; 12 and13; 14 and 15; 79 and 3; 80 and 4; 80 and 5; 80 and 6; 80 and 7; 80 and3; 82 and 11; 83 and 11; 84 and 11; 85 and 13; and 86 and 15;respectively. In some embodiments, the antibody or antigen-bindingfragment thereof binds to the same epitope of human ILT4 as an antibodyor antigen-binding fragment thereof comprising the heavy chain variabledomain and light chain variable domain amino acid sequences set forth inSEQ ID NOs:63 and 70; 57 and 71; 57 and 72; 57 and 73; 57 and 58; 57 and70; 64 and 74; 65 and 74; 66 and 74; 67 and 75; 68 and 76; respectively.

The present invention further provides an antibody or antigen-bindingfragment thereof that competes for binding to human ILT4 with anantibody or antigen-binding fragment thereof disclosed herein. Incertain embodiments, the antibody or antigen-binding fragment thereofcompetes for binding to human ILT4 with an antibody or fragmentcomprising the heavy chain and light chain amino acid sequences setforth in SEQ ID NOs:1 and 3; 2 and 4; 2 and 5; 2 and 6; 2 and 7; 2 and3; 8 and 11; 9 and 11; 10 and 11; 12 and 13; 14 and 15; 79 and 3; 80 and4; 80 and 5; 80 and 6; 80 and 7; 80 and 3; 82 and 11; 83 and 11; 84 and11; 85 and 13; and 86 and 15; respectively. In some embodiments, theantibody or antigen-binding fragment thereof competes for binding tohuman ILT4 with an antibody or fragment comprising the heavy chainvariable domain and light chain variable domain amino acid sequences setforth in SEQ ID NOs:63 and 70; 57 and 71; 57 and 72; 57 and 73; 57 and58; 57 and 70; 64 and 74; 65 and 74; 66 and 74; 67 and 75; 68 and 76;respectively.

In addition, the present invention provides an antibody orantigen-binding fragment thereof that binds human ILT4, comprising: (a)the complementarity determining region-L1 (CDR-L1), complementaritydetermining region-L2 (CDR-L2), and complementarity determiningregion-L3 (CDR-L3) of a light chain variable (V_(L)) domain of animmunoglobulin chain that comprises the amino acid sequence set forth inSEQ ID NO: 3-7, 11, 13, 15, or 45; and/or (b) the complementaritydetermining region-H1 (CDR-H1), complementarity determining region-H2(CDR-H2), and complementarity determining region-H3 (CDR-H3) of a heavychain variable (V_(H)) domain of an immunoglobulin chain that comprisesthe amino acid sequence set forth in SEQ ID NO: 1, 2, 8-10, 12, 14, 44,or 79-86.

In an embodiment of the invention, the antibody or antigen-bindingfragment thereof comprises: (1) a V_(H) domain comprising: CDR-H1: GYYWS(SEQ ID NO: 16), CDR-H2: EINHXGSTNYNPSLKS wherein X is S or A (SEQ IDNO: 17); and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GX₁X₂NRPS, wherein X₁ is S or A and X₂ is N, Q, E or D (SEQ ID NO: 20);and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21); (2) a V_(H) domain comprising:CDR-H1: SYAIS (SEQ ID NO: 22); CDR-H2: GIIPIFGTANYAQKFQG (SEQ ID NO:23); and CDR-H3: YFX₁X₂SGWYKGGAFDI, wherein X₁ is D or S and X₂ is S orA (SEQ ID NO: 24); and/or, a V_(L) domain comprising: CDR-L1:TLRSGINVDTYRIH (SEQ ID NO: 25); CDR-L2: YKSDSDKHQGS (SEQ ID NO: 26); andCDR-L3: AIWYSSTWV (SEQ ID NO: 27); (3) a V_(H) domain comprising:CDR-H1: SYAMH (SEQ ID NO: 28); CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO:29); and CDR-H3: VGEWIQLWSPFDY (SEQ ID NO: 30); and/or, a V_(L) domaincomprising: CDR-L1: RASQGISSWLA (SEQ ID NO: 31); CDR-L2: AASSLQS (SEQ IDNO: 32); and CDR-L3: QQYNSYPPT (SEQ ID NO: 33); and/or (4) a V_(H)domain comprising: CDR-H1: ELSMH (SEQ ID NO: 34); CDR-H2:GFDPEDGETIYAQKFQG (SEQ ID NO: 35); and CDR-H3: AGPLYTIFGVVIIPDNWFDP (SEQID NO: 36); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 37); CDR-L2: GNSNRPS (SEQ ID NO: 38); and CDR-L3:QSYDSSLSGSGVV (SEQ ID NO: 39).

In one embodiment, the antibody or antigen-binding fragment comprises: aV_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2:EINHXGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ IDNO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ IDNO: 21).

In another embodiment, the antibody or antigen-binding fragmentcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises a light chain immunoglobulin, a heavy chain immunoglobulin, orboth a light and heavy chain immunoglobulin, wherein the light chainimmunoglobulin has at least 90% amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 11, 13, 15, or45, and/or the heavy chain immunoglobulin has at least 90% amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:1,2, 8, 9, 10, 12, 14, 44, 79, 80, 81, 82, 83, 84, 85, or 86.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises a light chain immunoglobulin, a heavy chain immunoglobulin, orboth a light and heavy chain immunoglobulin, wherein the light chainimmunoglobulin comprises a light chain variable domain having at least90% amino acid sequence identity to the amino acid sequence set forth inSEQ ID NO:70, 71, 72, 73, 58, 74, 75, 76, or 77, and/or the heavy chainimmunoglobulin comprises a heavy chain variable domain having at least90% amino acid sequence identity to the amino acid sequence set forth inSEQ ID NO:63, 57, 64, 65, 66, 67, 68, or 69.

In still other embodiments, the antibody or antigen-binding fragmentthereof comprises a light chain immunoglobulin, a heavy chainimmunoglobulin, or both a light and heavy chain immunoglobulin, whereinthe light chain immunoglobulin comprises the amino acid sequence setforth in SEQ ID NO:3, 4, 5, 6, 7, 11, 13, 15, or 45; and/or the heavychain immunoglobulin comprises the amino acid sequence set forth in SEQID NO: 1, 2, 8, 9, 10, 12, 14, 44, 79, 80, 81, 82, 83, 84, 85, or 86.

In yet still other embodiments, the antibody or antigen-binding fragmentthereof comprises a light chain immunoglobulin, a heavy chainimmunoglobulin, or both a light and heavy chain immunoglobulin, whereinthe light chain variable domain comprises the amino acid sequence setforth in SEQ ID NO:70, 71, 72, 73, 58, 74, 75, 76, or 77, and/or theheavy chain variable domain comprise the amino acid sequence set forthin SEQ ID NO:63, 57, 64, 65, 66, 67, 68, or 69.

Further provided is an antibody or antigen-binding fragment thereofcomprising any of the following sets of heavy chain immunoglobulins andlight chain immunoglobulins: (1) a heavy chain immunoglobulin comprisingthe amino acid sequence set forth in SEQ ID NO:1; a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:3; (2) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:2; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:4; (3) a heavychain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:2; a light chain immunoglobulin comprising the amino acid sequenceset forth in SEQ ID NO:5; (4) a heavy chain immunoglobulin comprisingthe amino acid sequence set forth in SEQ ID NO:2; a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:6; (5) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:2; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:7; (6) a heavychain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:2; a light chain immunoglobulin comprising the amino acid sequenceset forth in SEQ ID NO:3; (7) a heavy chain immunoglobulin comprisingthe amino acid sequence set forth in SEQ ID NO:8; a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:11; (8) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:9; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:11; (9) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:10; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:11; (10) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:12; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:13; (11) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:14; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:15; (12) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:79; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:3; (13) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:80; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:4; (14) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:80; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:5; (15) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:80; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:6; (16) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:80; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:7; (17) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:80; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:3; (18) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:82; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:11; (19) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:83; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:11; (20) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:84; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:11; (21) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:85; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:13; or (22) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:86; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:15.

In addition, provided herein is an antibody or antigen-binding fragmentthereof comprising any of the following sets of heavy chain variabledomain and light chain variable domain: (1) a heavy chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:63; anda light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:70; (2) a heavy chain variable domain comprising theamino acid sequence set forth in SEQ ID NO:57; and a light chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO:71; (3) a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:57; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:72; (4) aheavy chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:57; and a light chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:73; (5) a heavy chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:57; anda light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:58; (6) a heavy chain variable domain comprising theamino acid sequence set forth in SEQ ID NO:57; and a light chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO:70; (7) a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:64; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:74; (8) aheavy chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:65; and a light chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:74; (9) a heavy chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:66; anda light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:74; (10) a heavy chain variable domain comprising theamino acid sequence set forth in SEQ ID NO:67; and a light chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO:75; or (11) a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:68; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:76.

In one preferred embodiment, the antibody or antigen-binding fragmentthereof comprises a V_(H) domain comprising the amino acid sequence setforth in SEQ ID NO:57; and a V_(L) domain comprising the amino acidsequence set forth in SEQ ID NO:58.

In another preferred embodiment, the antibody or antigen-bindingfragment thereof comprises: a heavy chain immunoglobulin comprising theamino acid sequence set forth in SEQ ID NO:2; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:7.

In yet another preferred embodiment, the antibody or antigen-bindingfragment thereof comprises: a heavy chain immunoglobulin comprising theamino acid sequence set forth in SEQ ID NO:80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:7.

In one embodiment, the antibody consists of two heavy chains and twolight chains, wherein each light chain comprises the amino acid sequenceset forth in SEQ ID NO:58 and each heavy chain comprises the amino acidsequence set forth in SEQ ID NO:57.

In another embodiment, the antibody consists of two heavy chains and twolight chains, wherein each light chain comprises the amino acid sequenceset forth in SEQ ID NO:58 and each heavy chain comprises the amino acidsequence set forth in SEQ ID NO:57, wherein the light chain furthercomprises the amino acid sequence set forth in SEQ ID NO:90.

In yet another embodiment, the antibody consists of two heavy chains andtwo light chains, wherein each light chain comprises the amino acidsequence set forth in SEQ ID NO:58 and each heavy chain comprises theamino acid sequence set forth in SEQ ID NO:57, wherein the heavy chainfurther comprises the amino acid sequence set forth in SEQ ID NO:89.

In still another embodiment, the antibody consists of two heavy chainsand two light chains, wherein each light chain comprises the amino acidsequence set forth in SEQ ID NO:58 and each heavy chain comprises theamino acid sequence set forth in SEQ ID NO:57, wherein the light chainfurther comprises the amino acid sequence set forth in SEQ ID NO:90 andthe heavy chain further comprises the amino acid sequence set forth inSEQ ID NO:89.

In one embodiment, the antibody consists of two heavy chains and twolight chains, wherein each light chain comprises the amino acid sequenceset forth in SEQ ID NO:7 and each heavy chain comprises the amino acidsequence set forth in SEQ ID NO:2.

In another embodiment, the antibody consists of two heavy chains and twolight chains, wherein each light chain consists of the amino acidsequence set forth in SEQ ID NO:7 and each heavy chain consists of theamino acid sequence set forth in SEQ ID NO:2.

In an embodiment of the invention, the antibody or antigen-bindingfragment thereof is glycosylated, e.g., with engineered yeast N-linkedglycans or Chinese hamster ovary (CHO) cell N-linked glycans. In anembodiment of the invention, the antibody or antigen-binding fragmentthereof is an antibody.

The present invention also provides a pharmaceutical compositioncomprising an antibody or antigen-binding fragment thereof disclosedherein. In certain embodiments, the composition further comprises atherapeutic agent (e.g., pembrolizumab). In some embodiments, thecomposition further comprises a pharmaceutically acceptable carrier. Inother embodiments, the composition further comprises a therapeutic agent(e.g., pembrolizumab) and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition comprises: (i) anantibody that consists of two heavy chains and two light chains, whereineach light chain consists of the amino acid sequence set forth in SEQ IDNO:7 and each heavy chain consists of the amino acid sequence set forthin SEQ ID NO:2, and (ii) pembrolizumab.

The present invention further provides a polypeptide (e.g., an isolatedpolypeptide) which includes the immunoglobulin light chain and/orimmunoglobulin heavy chain or a variable domain thereof of any antibodyor antigen-binding fragment thereof disclosed herein. For example, in anembodiment of the invention, the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-39, 44, 45,47-58, 63-77, and 79-86. Also provided by the present invention is anypolynucleotide (e.g., DNA or RNA) that encodes any polypeptide disclosedherein. In another aspect, provided is a vector comprising thepolynucleotide disclosed herein. The present invention also provides ahost cell (e.g., a CHO cell) comprising the polynucleotide or the vectordisclosed herein.

The present invention provides a method for blocking binding of ILT4 toHLA-G, HLA-A, HLA-B, and/or HLA-F, e.g., in vitro or in vivo, forexample, in the body of a subject (e.g., a human subject) in needthereof, comprising administering to the subject an effective amount ofthe antibody or antigen-binding fragment thereof disclosed herein. Incertain embodiments, the method for blocking binding of ILT4 to HLA-G,HLA-A, HLA-B, and/or HLA-F further comprises performing a therapeuticprocedure (e.g., anti-cancer radiation therapy or surgical tumorectomy)to the subject. In some embodiments, the method for blocking binding ofILT4 to HLA-G, HLA-A, HLA-B, and/or HLA-F further comprisesadministering a therapeutic agent (e.g., pembrolizumab) to the subject.In other embodiments, the method for blocking binding of ILT4 to HLA-G,HLA-A, HLA-B, and/or HLA-F further comprises performing a therapeuticprocedure (e.g., anti-cancer radiation therapy or surgical tumorectomy)and administering a therapeutic agent (e.g., pembrolizumab) to thesubject.

The present invention also provides a method of treating a cancer in asubject (e.g., a human subject), comprising administering to the subjectan effective amount of the antibody or antigen-binding fragment thereofdisclosed herein. In certain embodiments, the method of treating acancer further comprises performing a therapeutic procedure (e.g.,anti-cancer radiation therapy or surgical tumorectomy) to the subject.In some embodiments, the method of treating a cancer further comprisesadministering a therapeutic agent (e.g., pembrolizumab) to the subject.In other embodiments, the method of treating a cancer further comprisesperforming a therapeutic procedure (e.g., anti-cancer radiation therapyor surgical tumorectomy) and administering a therapeutic agent (e.g.,pembrolizumab) to the subject.

The present invention also provides a method of producing the antibodyor antigen-binding fragment thereof of the present invention or animmunoglobulin chain thereof (e.g., a V_(H) and/or V_(L) thereof),comprising culturing a host cell (e.g., a CHO cell) comprising apolynucleotide (e.g., wherein the polynucleotide is in a vector and/oris integrated into one or more chromosomes of the host cell) encodingthe antibody or antigen-binding fragment thereof or an immunoglobulinchain thereof to express the antibody or antigen-binding fragmentthereof or an immunoglobulin chain thereof.

Also provided is a method of producing the antibody or antigen-bindingfragment thereof of the present invention or an immunoglobulin chainthereof (e.g., a V_(H) and/or V_(L) thereof), comprising: expressing apolynucleotide encoding the antibody or antigen-binding fragment thereofor an immunoglobulin chain thereof.

An antibody or antigen-binding fragment thereof that binds human ILT4 oran immunoglobulin chain thereof which is a product of said method isalso part of the present invention.

A method for detecting the presence of an ILT4 peptide or a fragmentthereof in a sample also forms part of the present invention. The methodcomprises contacting the sample with an antibody or antigen-bindingfragment of the present invention and detecting the presence of acomplex between the antibody or antigen-binding fragment and the ILT4peptide or fragment thereof, wherein detection of the complex indicatesthe presence of the ILT4 peptide or fragment thereof. In an embodimentof the invention, the method is performed in vitro, e.g., in abiological sample, e.g., surgical section or blood sample, of a subject.In another embodiment, the method is performed in vivo, e.g., in thebody of a subject. In yet another embodiment, the subject is a humanbeing.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Deuterium labeling Heatmap of p1E1(G1) binding to ILT4-His.

FIGS. 2A and 2B show the crystal structure of human ILT4. FIG. 2Adepicts the deuterium labeling levels mapped onto the structure of humanILT4. FIG. 2B shows the crystal structure of domains 1 and 2 of humanILT4 complexed with HLA-G. ILT4, HLA-G heavy chain, andbeta-2-microglobulin are indicated. The human ILT4 epitopes, havingresidues LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ IDNO:61), and HTGRYGCQ (SEQ ID NO:62), are indicated.

FIGS. 3A and 3B show ILT4 HLA-G binding and 1E1(G4) blockade. Mouse 3A9T cells transfected with human ILT4 were blocked with Fc block, thenincubated with titrated concentrations of 1E1(G4) (starting at 27 ug/mL,1:3 dilutions), or with hIgG4 isotype control (27 ug/mL). FIG. 3A.1E1(G4) or hIgG4 was detected with fluorochrome labeled goat anti-humanF(ab)₂ and detected by flow cytometry. FIG. 3B. Following 1E1(G4)pre-treatment, cells were incubated with 2 ug/mL biotinylated HLA-Fc orcontrol Fc (mVISTA-Fc). Fc binding was detected with PE conjugatedstreptavidin and detected by flow cytometry. Plots shown arerepresentative of 3 independent experiments. IC50 and EC50 values shownare the average of these experiments+/−standard deviation.

FIG. 4. Non-HLA-G MHC class I ligand binding to ILT4 and p1E1(G1)blockade. Mouse 3A9 T cells transfected with human ILT4 were pretreatedwith titrated concentrations of p1E1(G1) (starting at 10 ug/mL, 1:3dilutions), or with hIgG1 isotype control (10 ug/mL) before incubationwith fluorochrome labeled tetramers of HLA-F or CD1d, or fluorochromelabeled dexamers of HLA*A2:01 or HLA*B7:02. Tetramer/dexamer binding wasdetermined by flow cytometry and the mean fluorescence intensity of eachwas plotted. The dilutions/concentrations used for each dexamer/tetramerare as follows: HLA*A2:01-dex PE: 1:25; HLA*B7:02-dex FITC: 1:25;CD1d-tet PE: 1:50; HLA-F-tet PE: 1 ug/mL.

FIGS. 5A-5C. ANGPTL binding to ILT4 and p1E1(G1) blockade. FIG. 5A.Biotinylated ANGPTL proteins were preincubated for 20 min. with p1E1(G1)or human IgG1, the final concentration of each was 20 ug/mL. Solutionswere then added to mouse 3A9 T cells transfected with human ILT4 andwere incubated for an additional 30 minutes. ANGPTL binding was detectedwith PE conjugated streptavidin and analyzed by flow cytometry. PElabeled HLA-G tetramer was also added as a positive ILT4binding/blocking control. ANGPTL proteins were purchased from R&DSYSTEMS and biotinylated; FIG. 5B. Mouse 3A9 T cells transfected withhuman ILT4 were blocked with Fc block, then incubated with 20 ug/mL ofp1E1(G1) or hIgG1 isotype control. Following incubation, p1E1 (G1) orhIgG1 was detected with fluorochrome labeled goat anti-human F(ab′)2 andanalyzed by flow cytometry; FIG. 5C. Vector control 3A9 T-cells wereused as a negative control for ANGPTL binding. Cells were treated asdescribed in (A) except no treatment with antibody was performed.

FIGS. 6A and 6B. ILT family 1E1(G4) binding specificity. Mouse 3A9 Tcells transfected with human ILT family members derived from consensussequences published in the Uniprot database were used to test binding ofhIgG4 isotype control antibody or 1E1 (G4) at a fixed dose of 10 ug/mL.Vector control 3A9 T cells were used as an additional negative control.Also shown is binding of commercially available ILT-reactive antibodiescompared to their respective isotype control to demonstrate ILT-familymember expression. Data shown is representative of two experiments withsimilar results. See the legend embedded in the figure.

FIG. 7. Rescue of IL-2 release from ILT4 3A9 T cell transfectants with1E1(G4). Mouse 3A9 T cells transfected with human ILT4 were treated withplatebound anti-CD3 antibody in the presence of soluble 1E1(G4) orisotype control (huIgG4), starting at 27 ug/mL and serially diluted3-fold to 0.3 ug/mL. After 24 hrs of incubation, supernatants areremoved and mouse IL-2 is measured by ELISA. Plot shown isrepresentative of 5 independent experiments. EC50 value shown is theaverage of these experiments+/−standard deviation.

FIG. 8. p1E1(G4) and 1E1(G4) rescued ILT4:HLA-G induced suppression ofmast cell degranulation. Mouse WTMC mast cells were transfected withhuman ILT4 and pretreated with titrated concentrations of 1E1(G4),p1E1(G4), or hIgG4 isotype control (starting at 10 ug/mL, 1:3 dilutions)before stimulating with platebound anti-CD200Rla (Clone DX89; 1 ug/mL)and platebound HLA-G tetramer (0.625 ug/mL). Following stimulation for 1hour, degranulation was assessed by collecting supernatants from themast cells to measure the release of β-hexoseaminidase using acolorimetric enzymatic assay. Data shown is representative of 2independent experiments with 3 technical replicates per data point.

FIGS. 9A and 9B. 1E1(G4) enhanced LPS-induced expression ofpro-inflammatory myeloid cytokines. Whole PBMCs from healthy patientswere isolated from leukoreduction chambers and treated with 0.25 ug/mLLPS in the presence of either hIgG4 (30 ug/mL; open circles) or 1E1(G4)(marked as “1E1” in figure) (between 30 ug/mL and 3 pg/mL; closedcircles) for 3 days. Following stimulation, supernatants were assayedfor cytokine expression ((FIG. 9A) GM-CSF and (FIG. 9B) TNFa) using aMeso Scale Discovery multi-cytokine assay kit. Each color representsdata from an individual patient. Conditions without any stimulation arealso shown (closed triangles).

FIGS. 10A and 10B. 1E1(G4) enhanced anti-CD3-induced expression ofpro-inflammatory myeloid cytokines. (A-B) Whole PBMCs from healthypatients were isolated from leukoreduction chambers and treated with0.01 ug/mL anti-CD3 (HIT3a) in the presence of either hIgG4 (30 ug/mL;open circles) or 1E1(G4) (marked as “1E1” in figure) (between 30 ug/mLand 3 pg/mL; closed circles) for 3 days. Following stimulation,supernatants were assayed for cytokine expression ((FIG. 10A) GM-CSF and(FIG. 10B) TNFa) using a Meso Scale Discovery multi-cytokine assay kit.Each color represents data from an individual patient. Conditionswithout any stimulation are also shown (closed triangles).

FIGS. 11A-11E. p1E1(G4) treatment leads to tumor growth inhibition in ahumanized mouse SKMEL5 tumor model. CD34+Cord blood-engrafted humanizedNSG mice, from 2 different cord blood donors, were subcutaneouslyinoculated with 1×10⁶ SKMEL5 tumor cells in their left flanks. Followinginoculation, tumors were allowed to grow and those which reached anaverage size of 150 mm³ were randomized into groups of 6 (3 from eachstem cell donor, n=6 per group total). Mice were then challenged witheither hIgG4 isotype control or p1E1(G4) (20 mgs/kg each) every 5 days,with tumors and weights measured weekly, until the end of the study.Tumor growth in the isotype control and p1E1(G4) treated mice weretracked over time. FIG. 11A shows mean tumor volume (mm³)+/−SD over timefor both groups, and FIGS. 11B and 11C show individual mouse tumorvolumes (mm³) over time for isotype treated and p1E1(G4), respectively.FIG. 11D shows tumor weight in individual mice treated with isotypecontrol or p1E1(G4) as followed over time; FIG. 11E shows weight loss ofeach treatment group was also measured over time. Following studycompletion, mice were sacrificed and tumors were harvested and weighed.

FIGS. 12A-12D demonstrate that 1E1(G4) treatment led to tumor growthinhibition in a humanized mouse SK-MEL-5 tumor model. FIG. 12A showsmean tumor volume (mm³)+/−SD over time for both 1E1(G4)-treated mice andIgG4 isotype control-treated mice. FIG. 12B shows body weight changeover time for both groups. FIG. 12C shows endpoint tumor weight inindividual mice treated with isotype control or 1E1(G4). FIG. 12D showsendpoint spleen weight in individual mice treated with isotype controlor 1E1(G4).

FIG. 13. ILT4 haplotype binding. Mouse 3A9 T cells transfected withhuman ILT4 allelic variants were used to test binding of hIgG4 isotypecontrol antibody or 1E1(G4) at a fixed dose of 10 ug/mL. Vector control3A9 T cells were used as an additional negative control. Haplotypes areexplained in Table 2. Data shown is representative of two experimentswith similar results.

FIGS. 14A and 14B. ILT4 RNA expression in different tumor types or celltypes according to public databases. FIG. 14A depicts ILT4 RNAexpression in various tumor types according to the TOGA database. FIG.14B depicts ILT4 RNA expression in various cell types according to theBlueprint database.

FIGS. 15A and 15B show 1E1(G4) binding to myeloid cells from renal cellcarcinoma (RCC) (FIG. 15A) and colorectal cancer (CRC) (FIG. 15B) tumorhistoculture samples.

FIG. 16. Predominant N-linked glycans for monoclonal antibodies producedin Chinese hamster ovary cells (CHO N-linked glycans) and in engineeredyeast cells (engineered yeast N-linked glycans): squares:N-acetylglucosamine (GlcNac); circles: mannose (Man); diamonds:galactose (Gal); triangles: fucose (Fuc).

FIG. 17 shows anti-tumor efficacy of various anti-ILT4 antibodies in ahumanized mouse SK-MEL-5 tumor model.

DETAILED DESCRIPTION OF THE INVENTION

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)). Affinity can be measured by common methods known inthe art, including KinExA and Biacore.

As used herein, the term “antibody” includes, but is not limited to,monoclonal antibodies, polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), fully human antibodies, and chimericantibodies.

As used herein, unless otherwise indicated, “antigen-binding fragment”refers to antigen-binding fragments of antibodies, i.e. antibodyfragments that retain the ability to bind to the antigen bound by thefull-length antibody, e.g. fragments that retain one or more CDRregions. Examples of antibody binding fragments include, but are notlimited to, Fab, Fab′, F(ab′)₂, Fv fragments and individual antibodyheavy chains or light chains, and individual heavy chain or light chainvariable regions.

A “Fab fragment” is comprised of one light chain and the CH1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule. An “Fabfragment” can be the product of papain cleavage of an antibody.

An “Fc” region contains two heavy chain fragments comprising the CH1 andCH2 domains of an antibody. The two heavy chain fragments are heldtogether by two or more disulfide bonds and by hydrophobic interactionsof the CH3 domains.

A “Fab′ fragment” contains one light chain and a portion or fragment ofone heavy chain that contains the V_(H) domain and the CH1 domain andalso the region between the CH1 and CH2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the CH1 and CH2domains, such that an interchain disulfide bond is formed between thetwo heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains. An “F(ab′)₂ fragment” can be the product of pepsincleavage of an antibody.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

“Isolated antibody” refers to the purification status and in suchcontext means the molecule is substantially free of other biologicalmolecules such as nucleic acids, proteins, lipids, carbohydrates, orother material such as cellular debris and growth media. Generally, theterm “isolated” is not intended to refer to a complete absence of suchmaterial or to an absence of water, buffers, or salts, unless they arepresent in amounts that substantially interfere with experimental ortherapeutic use of the binding compound as described herein.

The term “monoclonal antibody”, as used herein, refers to a populationof substantially homogeneous antibodies, i.e., the antibody moleculescomprising the population are identical in amino acid sequence exceptfor possible naturally occurring mutations that may be present in minoramounts. In contrast, conventional (polyclonal) antibody preparationstypically include a multitude of different antibodies having differentamino acid sequences in their variable domains that are often specificfor different epitopes. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al. (1975) Nature 256: 495, or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al. (1991) Nature 352: 624-628 andMarks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See alsoPresta (2005) J. Allergy Clin. Immunol. 116:731.

The term “fully human antibody” refers to an antibody that compriseshuman immunoglobulin protein sequences only. A fully human antibody maycontain murine carbohydrate chains if produced in a mouse, in a mousecell, or in a hybridoma derived from a mouse cell. Similarly, “mouseantibody” refers to an antibody that comprises mouse immunoglobulinsequences only. Alternatively, a fully human antibody may contain ratcarbohydrate chains if produced in a rat, in a rat cell, or in ahybridoma derived from a rat cell. Similarly, “rat antibody” refers toan antibody that comprises rat immunoglobulin sequences only.

In general, the basic “antibody” structural unit comprises a tetramer.In an monospecific antibody, each tetramer includes two identical pairsof polypeptide chains, each pair having one “light” (about 25 kDa) andone “heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a “variable region” or “variable domain” of about 100 to110 or more amino acids primarily responsible for antigen recognition.The carboxy-terminal portion of the heavy chain may define a constantregion primarily responsible for effector function.

Typically, human constant light chains are classified as kappa andlambda light chains. Furthermore, human constant heavy chains aretypically classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Subtypes of these IgG include, for example, IgG1 and IgG4. The presentinvention includes anti-ILT4 antibodies and antigen-binding fragmentscomprising any of these light and/or heavy constant chains.

“Variable region,” “variable domain,” “V region,” or “V chain” as usedherein means the segment of IgG chains which is variable in sequencebetween different antibodies. A “variable region” of an antibody refersto the variable region of the antibody light chain or the variableregion of the antibody heavy chain, either alone or in combination. Thevariable region of the heavy chain may be referred to as “V_(H).” Thevariable region of the light chain may be referred to as “V_(L).”Typically, the variable regions of both the heavy and light chainscomprise three hypervariable regions, also called complementaritydetermining regions (CDRs), which are located within relativelyconserved framework regions (FR). The CDRs are usually aligned by theframework regions, enabling binding to a specific epitope. In general,from N-terminal to C-terminal, both light and heavy chains variabledomains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Theassignment of amino acids to each domain is, generally, in accordancewith the definitions of Sequences of Proteins of Immunological Interest,Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.;NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75;Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al.,(1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature342:878-883.

A “CDR” refers to one of three hypervariable regions (H1, H2, or H3)within the non-framework region of the antibody V_(H) β-sheet framework,or one of three hypervariable regions (L1, L2, or L3) within thenon-framework region of the antibody V_(L) β-sheet framework.Accordingly, CDRs are variable region sequences interspersed within theframework region sequences. CDR regions are well known to those skilledin the art and have been defined by, for example, Kabat as the regionsof most hypervariability within the antibody variable domains. CDRregion sequences also have been defined structurally by Chothia as thoseresidues that are not part of the conserved β-sheet framework, and thusare able to adapt to different conformation. Both terminologies are wellrecognized in the art. CDR region sequences have also been defined byAbM, Contact, and IMGT. The positions of CDRs within a canonicalantibody variable region have been determined by comparison of numerousstructures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-48; Morea etal., 2000, Methods 20:267-79). Because the number of residues within ahypervariable region varies in different antibodies, additional residuesrelative to the canonical positions are conventionally numbered with a,b, c and so forth next to the residue number in the canonical variableregion numbering scheme (Al-Lazikani et al., supra). Such nomenclatureis similarly well known to those skilled in the art. Correspondencebetween the numbering system, including, for example, the Kabatnumbering and the IMGT unique numbering system, is well known to oneskilled in the art and shown below in Table 1. In some embodiments, theCDRs are as defined by the Kabat numbering system. In other embodiments,the CDRs are as defined by the IMGT numbering system. In yet otherembodiments, the CDRs are as defined by the AbM numbering system. Instill other embodiments, the CDRs are as defined by the Chothianumbering system. In yet other embodiments, the CDRs are as defined bythe Contact numbering system.

TABLE 1 Correspondence between the CDR Numbering Systems Kabat + ChothiaIMGT Kabat AbM Chothia Contact V_(H) CDR1 26-35 27-38 31-35 26-35 26-3230-35 V_(H) CDR2 50-65 56-65 50-65 50-58 52-56 47-58 V_(H) CDR3  95-102105-117  95-102  95-102  95-102  93-101 V_(L) CDR1 24-34 27-38 24-3424-34 24-34 30-36 V_(L) CDR2 50-56 56-65 50-56 50-56 50-56 46-55 V_(L)CDR3 89-97 105-117 89-97 89-97 89-97 89-96

Sequence identity refers to the degree to which the amino acids of twopolypeptides are the same at equivalent positions when the two sequencesare optimally aligned.

Sequence similarity includes identical residues and non-identical,biochemically related amino acids. Biochemically related amino acidsthat share similar properties and may be interchangeable are discussedabove.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids in a protein with other amino acidshaving similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity of the protein. Those of skill in this art recognizethat, in general, single amino acid substitutions in non-essentialregions of a polypeptide do not substantially alter biological activity(see, e.g., Watson et al. (1987) Molecular Biology of the Gene, TheBenjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,substitutions of structurally or functionally similar amino acids areless likely to disrupt biological activity. Exemplary conservativesubstitutions are set forth in Table 2.

TABLE 2 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

The term “epitope,” as used herein, refers to an area or region on anantigen to which an antibody or antigen-binding fragment binds. Bindingof an antibody or antigen-binding fragment thereof disclosed herein toan epitope means that the antibody or antigen-binding fragment thereofbinds to one or more amino acid residues within the epitope.

“Isolated” nucleic acid molecule or polynucleotide means a DNA or RNA,e.g., of genomic, mRNA, cDNA, or synthetic origin or some combinationthereof which is not associated with all or a portion of apolynucleotide in which the isolated polynucleotide is found in nature,or is linked to a polynucleotide to which it is not linked in nature.For purposes of this disclosure, it should be understood that “apolynucleotide comprising” (or the like) a particular nucleotidesequence does not encompass intact chromosomes. Isolated polynucleotides“comprising” specified nucleic acid sequences may include, in additionto the specified sequences, coding sequences for up to ten or even up totwenty or more other proteins or portions or fragments thereof, or mayinclude operably linked regulatory sequences that control expression ofthe coding region of the recited nucleic acid sequences, and/or mayinclude vector sequences.

The phrase “control sequences” refers to polynucleotide sequencesnecessary or helpful for the expression of an operably linked codingsequence in a particular host organism. The control sequences that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. Eukaryotic cells areknown to use promoters, polyadenylation signals, and enhancers. In anembodiment of the invention, the polynucleotide is operably linked to apromoter such as a viral promoter, a CMV promoter, an SV40 promoter or anon-viral promoter or an elongation factor (EF)-1 promotor; and/or anintron.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another polynucleotide. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, but not always,“operably linked” means that the polynucleotide sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that not all progeny willhave precisely identical DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

Host cells include eukaryotic and prokaryotic host cells, includingmammalian cells. Host cells may be used as hosts for expression of theanti-ILT4 antibodies and antigen-binding fragments thereof. Host cellsinclude, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells,HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS),human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3cells and HEK-293 cells. Mammalian host cells include human, mouse, rat,dog, monkey, pig, goat, bovine, horse and hamster cells. Other celllines that may be used are insect cell lines (e.g., Spodopterafrugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plantcells and fungal cells. Fungal cells include yeast and filamentousfungus cells including, for example, Pichia pastoris, Pichia finlandica,Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichiaminuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum,Physcomitrella patens and Neurospora crassa. Pichia sp., anySaccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candidaalbicans, any Aspergillus sp., Trichoderma reesei, Chrysosporiumlucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurosporacrassa. The present invention includes any host cell (e.g., a CHO cellor Pichia cell, e.g., Pichia pastoris) containing an anti-ILT4 antibodyor antigen-binding fragment thereof or containing a polynucleotideencoding such an antibody or fragment or containing a vector thatcontains the polynucleotide.

“Treat” or “treating” means to administer anti-ILT4 antibodies orantigen-binding fragments thereof of the present invention, to a subjecthaving one or more symptoms of a disease for which the anti-ILT4antibodies and antigen-binding fragments are effective, e.g., in thetreatment of a subject having cancer or an infectious disease, or beingsuspected of having cancer or infectious disease, for which the agenthas therapeutic activity. Typically, the antibody or fragment isadministered in an “effective amount” or “effective dose” which willalleviate one or more symptoms (e.g., of cancer or infectious disease)in the treated subject or population, whether by inducing the regressionor elimination of such symptoms or by inhibiting the progression of suchsymptom(s), e.g., cancer symptoms such as tumor growth or metastasis, byany clinically measurable degree. The effective amount of the antibodyor fragment may vary according to factors such as the disease stage,age, and weight of the patient, and the ability of the drug to elicit adesired response in the subject.

As used herein, “an anti-ILT4 antibody or antigen-binding fragmentthereof” refers to an antibody or antigen-binding fragment thereof thatbinds to human ILT4.

The present invention includes antibodies and antigen-binding fragmentsthereof set forth herein that bind specifically to ILT4. An antibody orantigen-binding fragment binds “specifically” to a polypeptidecomprising a given sequence (e.g., human ILT4) if it binds topolypeptides comprising the sequence with a K_(D) of about 20 nM or ahigher affinity (e.g., about 17 nM, 10 nM, 5 nM, 1 nM, 100 pM, or 1 pM),but does not bind to proteins lacking the sequence. For example, anantibody or antigen-binding fragment that specifically binds to apolypeptide comprising human ILT4 may bind to a FLAG®-tagged form ofhuman ILT4 but will not bind to other FLAG®-tagged proteins that lackILT4 sequences.

ILT4

In an embodiment of the invention, the amino acid sequence of human ILT4comprises the amino acid sequence:

MTPIVTVLIC LGLSLGPRTH VQTGTIPKPT LWAEPDSVIT QGSPVTLSCQ GSLEAQEYRL  60YREKKSASWI TRIRPELVKN GQFHIPSITW EHTGRYGCQY YSRARWSELS DPLVLVMTGA 120YPKPTLSAQP SPVVTSGGRV TLQCESQVAF GGFILCKEGE EEHPQCLNSQ PHARGSSRAI 180FSVGPVSPNR RWSHRCYGYD LNSPYVWSSP SDLLELLVPG VSKKPSLSVQ PGPVVAPGES 240LTLQCVSDVG YDRFVLYKEG ERDLRQLPGR QPQAGLSQAN FTLGPVSRSY GGQYRCYGAH 300NLSSECSAPS DPLDILITGQ IRGTPFISVQ PGPTVASGEN VTLLCQSWRQ FHTFLLTKAG 360AADAPLRLRS IHEYPKYQAE FPMSPVTSAH AGTYRCYGSL NSDPYLLSHP SEPLELVVSG 420PSMGSSPPPT GPISTPAGPE DQPLTPTGSD PQSGLGRHLG VVIGILVAVV LLLLLLLLLF 480LILRHRRQGK HWTSTQRKAD FQHPAGAVGP EPTDRGLQWR SSPAADAQEE NLYAAVKDTQ 540PEDGVEMDTR AAASEAPQDV TYAQLHSLTL RRKATEPPPS QEREPPAEPS IYATLAIH 598(SEQ ID NO: 40, signal sequence underscored) See Uniprot accessionno. Q8N423.

In another embodiment of the invention, the amino acid sequence of humanILT4 comprises the following amino acid sequence without the signalsequence:

(SEQ ID NO: 78)                       QTGTIPKPT LWAEPDSVIT QGSPVTLSCQ GSLEAQEYRL  39YREKKSASWI TRIRPELVKN GQFHIPSITW EHTGRYGCQY YSRARWSELS DPLVLVMTGA  99YPKPTLSAQP SPVVTSGGRV TLQCESQVAF GGFILCKEGE EEHPQCLNSQ PHARGSSRAI 159FSVGPVSPNR RWSHRCYGYD LNSPYVWSSP SDLLELLVPG VSKKPSLSVQ PGPVVAPGES 219LTLQCVSDVG YDRFVLYKEG ERDLRQLPGR QPQAGLSQAN FTLGPVSRSY GGQYRCYGAH 279NLSSECSAPS DPLDILITGQ IRGTPFISVQ PGPTVASGEN VTLLCQSWRQ FHTFLLTKAG 339AADAPLRLRS IHEYPKYQAE FPMSPVTSAH AGTYRCYGSL NSDPYLLSHP SEPLELVVSG 399PSMGSSPPPT GPISTPAGPE DQPLTPTGSD PQSGLGRHLG VVIGILVAVV LLLLLLLLLF 459LILRHRRQGK HWTSTQRKAD FQHPAGAVGP EPTDRGLQWR SSPAADAQEE NLYAAVKDTQ 519PEDGVEMDTR AAASEAPQDV TYAQLHSLTL RRKATEPPPS QEREPPAEPS IYATLAIH  577.

In an embodiment of the invention, the amino acid sequence ofcynomolgous monkey ILT4 comprises the amino acid sequence:

MTPILMVLIC LGLSLGPRTH VQAGILPKPT LWAEPGSVIS EGSPVTLRCQ GSLQVQEYHL  60YREKNPASWV RQIRQELVKK GYFAIGFITW EHTGQYRCQY YSHSWWSEPS DPLELVVTGA 120YSKPTLSALP SPVVASGGNV TLQCDSQVAF DSFTLCKEGE DEHPQRLNCQ SHARGWSWAV 180FSVGPVSPSR RWSYRCYGYI SSAPNVWSLP SDLLELLVPG VSKKPSLSVQ PGPVVAPGDK 240LTLQCGSDAG YDRFALYKEG EGDFLQRPVR QPQAGLSQAN FLLGPVSRSH GGQYRCSGAH 300NLSSEWSAPS DPLDILIAGQ IRGRPFLSVQ PGPKVVSGEN VTLLCQSSWQ FHAFLLTQAG 360AADAHLHLRS MYKYPKYQAE FPMSPVTSAH AGTYRCYGSR SSNPYLLSVP SDPLELVVSG 420PSGGPSSPTT GPTSTCGPED QPLTPTGSAP QSGLGRHLGV VTGVLVAFVL LLFLLLLLFL 480VLRYRRQGKR WTSAQRKADF QHPAGAVEPE PRDRGLQRRS SPAADTQEEN LYAAVKDTQP 540EDGVELDSRA AASEDPQDVT YAQLQSLTLR REATEPPPSQ ERAPPVESSI YATLTIH 597(SEQ ID NO: 43, signal sequence underscored). See NCBI refseqXP_005590753.

In an embodiment of the invention, the signal sequence for expression ofILT4 or any other polypeptide set forth herein is MTPILMVLICLGLSLGPRTHV(amino acids 1-21 of SEQ ID NO:40) or MTPIVTVLICLGLSLGPRTHV (amino acids1-21 of SEQ ID NO:43) or MPLLLLLPLLWAGALA (SEQ ID NO:46).

In an embodiment of the invention, an anti-ILT4 antibody orantigen-binding fragment thereof of the present invention binds to theextracellular domain of ILT4:

QTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGV(amino acids 22-461 of SEQ ID NO: 40)or an immunoglobulin-fusion thereof (e.g., IgG1 or IgG4) or a cellsurface transmembrane (TM) form which is expressed on the surface of acell:

QTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVL LLLLLLLLFLILRHRRQGKH(amino acids 22-491 of SEQ ID NO: 40).

Antibodies and Antigen-Binding Fragments

The present invention provides antibodies and antigen-binding fragmentsthereof (e.g., fully human antibodies) that bind to ILT4 (hereinreferred to as “anti-ILT4”) and methods of use of the antibodies orantigen-binding fragments thereof in the treatment or prevention ofdisease. In one embodiment, the invention provides for antagonisticanti-ILT4 antibodies and methods of use of the antibodies orantigen-binding fragments thereof in the treatment or prevention ofdisease.

In one aspect, the present application includes anti-ILT4 antibodies andantigen-binding fragments thereof as set forth herein having one or moreof the properties set forth below:

-   -   binds human ILT4 at one or more amino acid residues in        LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID        NO:61), and/or HTGRYGCQ (SEQ ID NO:62), and/or protects        LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID        NO:61), and/or HTGRYGCQ (SEQ ID NO:62) from deuterium (e.g.,        D₂O) exchange, e.g., as determined by hydrogen-deuterium        exchange mass spectrometry and/or binds to ILT4 with a heat map        essentially as shown in FIG. 1;    -   binds human ILT4 at domain 1 (see Wilcox et al. BMC Structural        Biology 2:6 (2002));    -   binds human ILT4 extracellular domain or TM form of ILT4        expressed on a cell surface, e.g., a pre-B cell, Chinese hamster        ovary cell, U937 cell, or Jurkat JE6 cell.    -   calculated pI ˜7.29 (e.g., 7.29 or 7.30);    -   experimentally determined pI ˜7.2;    -   is characterized by a thermogram having Tm onset >60° C.,        Tm1˜65.2° C. and Tm2 ˜78.8° C.;    -   binds human ILT4 with a K_(D) of about 1.7×10⁻⁸ M (e.g., as        determined by surface plasmon resonance, e.g., binding of        anti-ILT4 to polyhistidine tagged human ILT4);    -   Ka=5.5×10⁵ M⁻¹ s⁻¹ (e.g., as determined by surface plasmon        resonance, e.g., binding of anti-ILT4 to polyhistidine tagged        human ILT4);    -   Kd=9×10⁻³s⁻¹ (e.g., as determined by surface plasmon resonance,        e.g., binding of anti-ILT4 to polyhistidine tagged human ILT4);    -   blocks binding of HLA-G (e.g., Fc fused HLA-G) to human ILT4        (e.g., ILT4 on mouse 3A9 T cells transfected with and expressing        ILT4), e.g., with an IC₅₀ of about 0.25 micrograms/ml (±0.06        micrograms/ml), e.g., as determined by surface plasmon        resonance;    -   blocks binding of HLA-A, HLA-B (e.g., fluorochrome labeled        dexamers of HLA-A, such as HLA*A2:01 or HLA-B such as        HLA*B7:02), and/or HLA-F (e.g., fluorochrome labeled tetramers        of HLA-F) to ILT4 (e.g., ILT4 on mouse 3A9 T cells transfected        with and expressing ILT4), e.g., as determined by surface        plasmon resonance;    -   blocks ILT4 (e.g., ILT4 on mouse 3A9 T cells transfected with        and expressing ILT4), binding to ANGPTL1, ANGPTL4, and/or        ANGPTL7 (e.g., biotinylated ANGPTL proteins), e.g., as        determined by surface plasmon resonance;    -   does not bind to ILT2, ILT3, ILT5, LILRB5, LILRA1, LILRA2, ILT7,        ILT8, and/or ILT11,    -   reverses ILT4-mediated suppression of 1L2 in ILT4 transfected        3A9 cells, e.g., with an EC₅₀ of 0.43 micrograms/ml (±0.14        micrograms/ml);    -   rescues ILT4:HLA-G induced suppression of mast cell        degranulation (e.g., In the presence of plate-bound HLA-G        tetramer), for example, wherein the mast cells express ILT4 and        CD200RLa and are stimulated, for example, with antibody-mediated        cross-linking of CD200RLa,    -   enhances lipopolysaccharide (LPS)-induced expression of        proinflammatory myeloid cytokines, for example, GM-CSF and/or        TNFalpha, from a peripheral blood mononuclear cell (PBMC);    -   enhances anti-CD3-induced expression of pro-inflammatory myeloid        cytokines for example, GM-CSF and/or TNFalpha, from a peripheral        blood mononuclear cell (PBMC);    -   inhibits tumor growth in humans or, for example, in other        mammals such as mice (e.g., Immuno-deficient NSG mice) which        were reconstituted with human hematopoietic stem cells, for        example, which harbor peripheral human CD45+ immune cells, for        example, wherein the tumor is a human skin melanoma tumor such        as from the cell line SKMEL5;    -   relieves macrophage/myeloid-derived suppressor cell        (MDSC)-mediated tumor tolerance in the body of a subject (e.g.,        human subject) with a tumor;    -   does not bind to cynomolgous monkey ILT4 and/or mouse pirB;        and/or    -   stains CD14+human monocytes and/or CD11B+human granulocytes    -   binds to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or all 10)        of the human ILT4 haplotypes set forth in Table 7.

Antibody 1E1 (Q1E) Heavy Chain (IgG4) Heavy Chain

EVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWS WIRQPPGKGLEWIG E INHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR LPT RWVTTRYFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 1; variable domain underscored; CDRs double underscored)

Heavy Chain Variable Domain

(SEQ ID NO: 63) EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPT RWVTTRYFDLWGRGTLVTVSS

Antibody 1E1 (Q1E, S54A) Heavy Chain (IgG4) Heavy Chain

EVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWS WIRQPPGKGLEWIG E INHAGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR RWVTTRYFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 2; variable domain underscored; CDRs double underscored)

Heavy Chain Variable Domain

(SEQ ID NO: 57) EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHAGSTNYNPSLKSRVTISVDTSKNQSLKLSSVTAADTAVYYCARLPTR WVTTRYFDLWGRGTLVTVSS

Antibody 1E1 Heavy Chain (IgG1) Heavy Chain

QVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWS WIRQPPGKGLEWIG E INHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR LPT RWVTTRYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPG K(SEQ ID NO: 44, variable domain underscored; CDRs double underscored)

Heavy chain variable domain

(SEQ ID NO: 69) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPT RWVTTRYFDLWGRGTLVTVSS

1E1 Heavy Chain CDRs

CDR-H1: (SEQ ID NO: 16) GYYWS CDR-H2: (SEQ ID NO: 17)EINHXGSTNYNPSLKS wherein X is S or A (e.g.,EINHSGSTNYNPSLKS or EINHAGSTNYNPSLKS) CDR-H3: (SEQ ID NO: 18)LPTRWVTTRYFDL

Antibody 1E1 (Q1E) Light Chain (Lambda) Light Chain

ESVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH WYQQLPGTAPKLL IY GNSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYC QSFDNSLS AYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3; variabledomain underscored; CDRs double underscored)

Light Chain Variable Domain

(SEQ ID NO: 70) ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLS AYVFGGGTQLTVL

Antibody 1E1 (Q1E, S54A) Light Chain (Lambda) Light Chain

ESVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH WYQQLPGTAPKLL IY GNANRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYC QSFDNSLS AYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 4; variabledomain underscored; CDRs double underscored)

Light Chain Variable Domain

(SEQ ID NO: 71) ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNANRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLS AYVFGGGTQLTVL

Antibody 1E1 (Q1E, N53Q) Light Chain (Lambda) Light Chain

ESVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH WYQQLPGTAPKLL IY

GVPDRFSVSKSGASASLAITGLQAEDEADYYC QSFDNSLS AYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 5; variabledomain underscored; CDRs double underscored)

Light Chain Variable Domain

(SEQ ID NO: 72) ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGQSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLS AYVFGGGTQLTVL

Antibody 1E1 (Q1E, N53E) Light Chain (Lambda) Light Chain

ESVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH WYQQLPGTAPKLL IY

GVPDRFSVSKSGASASLAITGLQAEDEADYYC QSFDNSLS AYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 6; variabledomain underscored; CDRs double underscored)

Light Chain Variable Domain

(SEQ ID NO: 73) ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGESNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLS AYVFGGGTQLTVL

Antibody 1E1 (Q1E, N53D) Light Chain (Lambda) Light Chain

ESVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH WYQQLPGTAPKLL IY

GVPDRFSVSKSGASASLAITGLQAEDEADYYC QSFDNSLS AYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 7; variabledomain underscored; CDRs double underscored)

Light Chain Variable Domain

(SEQ ID NO: 58) ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLS AYVFGGGTQLTVL

Antibody 1E1 Light Chain (Lambda) Light Chain

QSVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH WYQQLPGTAPKLL IY GNSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYC QSFDNSLS AYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 45, variabledomain underscored; CDRs double underscored)

Light Chain Variable Domain

(SEQ ID NO: 77) QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLS AYVFGGGTQLTVL

1E1 Light Chain CDRs

CDR-L1: (SEQ ID NO: 19) TGSSSNIGAGYDVH CDR-L2: (SEQ ID NO: 20)GX₁X₂NRPS, wherein X₁ is N,Q,E or D and X₂ is S orA (e.g., GNSNRPS, GNANRPS, GQSNRPS, GESNRPS or GDSNRPS) CDR-L3:(SEQ ID NO: 21) QSFDNSLSAYV

Antibodies and antigen-binding fragments thereof including the 1E1 heavyand light chain CDRs or the 1E1 V_(H) and V_(L) or the 1E1 heavy chainand light chain (or a variant thereof, e.g., as set forth herein) may bereferred to as “1E1.”

Antibody 2A6 (Q1E) Heavy Chain (IgG4) Heavy Chain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR YFDSSGWYKGGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 8; variable domainunderscored; CDRs double underscored)

Heavy Chain Variable Domain

(SEQ ID NO: 64) EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFDSSGWYKGGAFDIWGQGTMVTVSS

Antibody 2A6 (Q1E, S102A, M119L) Heavy Chain (IgG4) Heavy Chain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR

WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9; variable domainunderscored; CDRs double underscored)

Heavy Chain Variable Domain

(SEQ ID NO: 65) EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFDASGWYKGGAFDIWGQGTLVTVSS

Antibody 2A6 (Q1E, D101S, M119L) Heavy Chain (IgG4) Heavy Chain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR

WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10, variable domainunderscored; CDRs double underscored)

Heavy Chain Variable Domain

(SEQ ID NO: 66) EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFSSSGWYKGGAFDIWGQGTLVTVSS

The present invention includes antibodies and antigen-binding fragmentsthereof wherein residue 1 of SEQ ID NO:8, 9, 10, 64, 65, or 66 is Qinstead of E.

2A6 Heavy Chain CDRs

CDR-H1: (SEQ ID NO: 22) SYAIS CDR-H2: (SEQ ID NO: 23) GIIPIFGTANYAQKFQGCDR-H3: (SEQ ID NO: 24) YFX₁X₂SGWYKGGAFDI, wherein X₁ is D or S and X₂is S or A (e.g., YFDSSGWYKGGAFDI, YFDASGWYKGGAFDI or YFSSSGWYKGGAFDI)

Antibody 2A6 Light Chain (Lambda) Light Chain

QSVLTQPSSLSASPGASASLTC TLRSGINVDTYRIH WYQQKPGSPPQYL LR YKSDSDKHQGSGVPSRFSGSKDPSANAGILLISGLQSEDEADYYCAI WYSSTWVFGGGTQLTVLGQPKAAPSVTLEPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 11,variable domain underscored; CDRs double underscored)

Light Chain Variable Domain

(SEQ ID NO: 74) QSVLTQPSSLSASPGASASLTCTLRSGINVDTYRIHWYQQKPGSPPQYLLRYKSDSDKHQGSGVPSRFSGSKDPSANAGILLISGLQSEDEADYYCAI WYSSTWVFGGGTQLTVL

The present invention includes antibodies and antigen-binding fragmentsthereof wherein residue 1 of SEQ ID NO:11 or 74 is E instead of Q.

2A6 Light Chain CDRs

CDR-L1: (SEQ ID NO: 25) TLRSGINVDTYRIH CDR-L2: (SEQ ID NO: 26)YKSDSDKHQGS CDR-L3: (SEQ ID NO: 27) AIWYSSTWV

Antibodies and antigen-binding fragments thereof including the 2A6 heavyand light chain CDRs or the 2A6 V_(H) and V_(L) or the 2A6 heavy chainand light chain (or a variant thereof, e.g., as set forth herein) may bereferred to as “2A6.”

Antibody 3G7 (Q1E) Heavy Chain (IgG4) Heavy Chain

EVQLVESGGGVVQPGRSLRLSCAASGETFS SYAMH WVRQAPGKGLEWV A VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AR VGEWIQLWSPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 12, variabledomain underscored; CDRs double underscored).

Heavy Chain Variable Domain

(SEQ ID NO: 67) EVQLVESGGGVVQPGRSLRLSCAASGETFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGEWIQLWSPFDYWGQGTLVTVSS

The present invention includes antibodies and antigen-binding fragmentsthereof wherein residue 1 of SEQ ID NO:12 or 67 is Q instead of E.

3G7 Heavy Chain CDRs

CDR-H1: (SEQ ID NO: 28) SYAMH CDR-H2: (SEQ ID NO: 29) VISYDGSNKYYADSVKGCDR-H3: (SEQ ID NO: 30) VGEWIQLWSPFDY

Antibody 3G7 Light Chain (Kappa) Light Chain

DIQMTQSPSSVSASVGDRVTITC RASQGISSWLA WYQQKPGKAPKFLI Y AASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYC QQYNSYPP TFGGGTKVEIKRtVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13, variabledomain underscored; CDRs double underscored)

Light Chain Variable Domain

(SEQ ID NO: 75) DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKFLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPP TFGGGTKVEIK

3G7 Light Chain CDRs

CDR-L1: (SEQ ID NO: 31) RASQGISSWLA CDR-L2: (SEQ ID NO: 32) AASSLQSCDR-L3: (SEQ ID NO: 33) QQYNSYPPT

Antibodies and antigen-binding fragments thereof including the 3G7 heavyand light chain CDRs or the 3G7 V_(H) and V_(L) or the 3G7 heavy chainand light chain (or a variant thereof, e.g., as set forth herein) may bereferred to as “3G7.”

Antibody 2C1 (Q1E) Heavy Chain (IgG4) Heavy Chain

EVQLVQSGAEVKKPGASVKVSCKVSGYTLT ELSMH WVRQAPGKGLEWMG GFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAR AG PLYTIFGVVIIPDNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 14, variable domain under-scored; CDRs double underscored)

Heavy Chain Variable Domain

(SEQ ID NO: 68) EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARAGPLYTIFGVVIIPDNWFDPWGQGTLVTVSS

The present invention includes antibodies and antigen-binding fragmentsthereof wherein residue 1 of SEQ ID NO:14 or 68 is Q instead of E.

2C1 Heavy Chain CDRs

(SEQ ID NO: 34) CDR-H1: ELSMH (SEQ ID NO: 35) CDR-H2: GFDPEDGETIYAQKFQG(SEQ ID NO: 36) CDR-H3: AGPLYTIFGVVIIPDNWFDP

Antibody 2C1 Light Chain (Q1E) (Lambda) Light Chain

ESVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH WYQQLPGTAPKLLI Y GNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYC QSYDSSLSGS GVVFGGGTQLIILGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 15, variabledomain underscored; CDRs double underscored)

Light Chain Variable Domain

(SEQ ID NO: 76) ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGS GVVFGGGTQLIIL

The present invention includes antibodies and antigen-binding fragmentsthereof wherein residue 1 of SEQ ID NO:15 or 76 is Q instead of E.

2C1 Light Chain CDRs

CDR-L1: (SEQ ID NO: 37) TGSSSNIGAGYDVH CDR-L2: (SEQ ID NO: 38) GNSNRPSCDR-L3: (SEQ ID NO: 39) QSYDSSLSGSGVV

Antibodies and antigen-binding fragments thereof including the 2C1 heavyand light chain CDRs or the 2C1 V_(H) and V_(L) or the 2C1 heavy chainand light chain (or a variant thereof, e.g., as set forth herein) may bereferred to as “2C1.”

In various embodiments of the antibody or antigen-binding fragmentthereof, a C-terminal lysine of a heavy chain immunoglobulin is absent.

Thus, in some embodiments, the antibody or antigen-binding fragmentthereof comprises a light chain immunoglobulin, a heavy chainimmunoglobulin, or both a light and heavy chain immunoglobulin, whereinthe light chain immunoglobulin comprises the amino acid sequence setforth in SEQ ID NO:3, 4, 5, 6, 7, 11, 13, 15, or 45; and/or the heavychain immunoglobulin comprises the amino acid sequence set forth in SEQID NO: 1, 2, 8, 9, 10, 12, 14, 44, 79, 80, 81, 82, 83, 84, 85, or 86.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 1 or 79; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 3.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 2 or 80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 4.

In other embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 2 or 80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 5.

In yet other embodiments, the antibody or antigen-binding fragmentthereof comprises: a heavy chain immunoglobulin comprising the aminoacid sequence set forth in SEQ ID NO: 2 or 80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 6.

In still other embodiments, the antibody or antigen-binding fragmentthereof comprises: a heavy chain immunoglobulin comprising the aminoacid sequence set forth in SEQ ID NO: 2 or 80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 7.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 2 or 80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 3.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 8 or 82; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 11.

In other embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 9 or 83; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 11.

In yet other embodiments, the antibody or antigen-binding fragmentthereof comprises: a heavy chain immunoglobulin comprising the aminoacid sequence set forth in SEQ ID NO: 10 or 84; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 11.

In still other embodiments, the antibody or antigen-binding fragmentthereof comprises: a heavy chain immunoglobulin comprising the aminoacid sequence set forth in SEQ ID NO: 12 or 85; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 13.

In yet still embodiments, the antibody or antigen-binding fragmentthereof comprises: a heavy chain immunoglobulin comprising the aminoacid sequence set forth in SEQ ID NO: 14 or 86; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 15.

In certain other embodiments, the antibody or antigen-binding fragmentthereof comprises a light chain immunoglobulin, a heavy chainimmunoglobulin, or both a light and heavy chain immunoglobulin, whereinthe light chain variable domain comprises the amino acid sequence setforth in SEQ ID NO:70, 71, 72, 73, 58, 74, 75, 76, or 77, and/or theheavy chain variable domain comprise the amino acid sequence set forthin SEQ ID NO:63, 57, 64, 65, 66, 67, 68, or 69.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:63; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:70.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:57; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:71.

In other embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:57; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:72.

In yet other embodiments, the antibody or antigen-binding fragmentthereof comprises: a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:57; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:73.

In still other embodiments, the antibody or antigen-binding fragmentthereof comprises: a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:57; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:58.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:57; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:70.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises:

a heavy chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:64; and a light chain variable domain comprising theamino acid sequence set forth in SEQ ID NO:74.

In other embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:65; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:74.

In yet other embodiments, the antibody or antigen-binding fragmentthereof comprises: a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:66; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:74.

In still other embodiments, the antibody or antigen-binding fragmentthereof comprises: a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:67; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:75.

In yet still other embodiments, the antibody or antigen-binding fragmentthereof comprises: a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:68; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:76.

In a further embodiment, the antibody or antigen-binding fragmentthereof that binds ILT4 comprises an immunoglobulin light chain variable(V_(L)) domain comprising a CDR-L1, CDR-L2 and CDR-L3 of 1E1 (e.g., SEQID NOs: 19-21); and an immunoglobulin heavy chain variable (V_(H))domain comprising a CDR-H1, CDR-H2 and CDR-H3 of 1E1 (e.g., SEQ ID NOs:16-18).

In a further embodiment, the antibody or antigen-binding fragmentthereof that binds ILT4 comprises an immunoglobulin light chain variable(V_(L)) domain comprising a CDR-L1, CDR-L2 and CDR-L3 of 2A6 (e.g., SEQID NOs: 25-27); and an immunoglobulin heavy chain variable (V_(H))domain comprising a CDR-H1, CDR-H2 and CDR-H3 of 2A6 (e.g., SEQ ID NOs:22-24).

In a further embodiment, the antibody or antigen-binding fragmentthereof that binds ILT4 comprises an immunoglobulin light chain variable(V_(L)) domain comprising a CDR-L1, CDR-L2 and CDR-L3 of 3G7 (e.g., SEQID NOs: 31-33); and an immunoglobulin heavy chain variable (V_(H))domain comprising a CDR-H1, CDR-H2 and CDR-H3 of 3G7 (e.g., SEQ ID NOs:28-30).

In a further embodiment, the antibody or antigen-binding fragmentthereof that binds ILT4 comprises an immunoglobulin light chain variable(V_(L)) domain comprising a CDR-L1, CDR-L2 and CDR-L3 of 2C1 (e.g., SEQID NOs: 37-39); and an immunoglobulin heavy chain variable (V_(H))domain comprising a CDR-H1, CDR-H2 and CDR-H3 of 2C1 (e.g., SEQ ID NOs:34-36).

In one embodiment, the antibody or antigen-binding fragment comprises: aV_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2:EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ IDNO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ IDNO: 21).

In another embodiment, the antibody or antigen-binding fragmentcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereofcomprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16),CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQID NO: 18); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV(SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragmentthereof comprises: a V_(H) domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL(SEQ ID NO: 18); and/or, a V_(L) domain comprising: CDR-L1:TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), andCDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

The present invention further provides an antibody or antigen-bindingfragment thereof that binds ILT4 and comprises the V_(L) domain ofantibody 1E1 (e.g., SEQ ID NO:70, 71, 72, 73, 58 or 77) and/or the V_(H)domain of antibody 1E1 (e.g., SEQ ID NO:63, 57 or 69).

The present invention further provides an antibody or antigen-bindingfragment thereof that binds ILT4 and comprises the V_(L) domain ofantibody 2A6 (e.g., SEQ ID NO:74) and/or the V_(H) domain of antibody2A6 (e.g., SEQ ID NO:64, 65 or 66).

The present invention further provides an antibody or antigen-bindingfragment thereof that binds ILT4 and comprises the V_(L) domain ofantibody 3G7 (e.g., SEQ ID NO:75) and/or the V_(H) domain of antibody3G7 (e.g., SEQ ID NO:67).

The present invention further provides an antibody or antigen-bindingfragment thereof that binds ILT4 and comprises the V_(L) domain ofantibody 2C1 (e.g., SEQ ID NO:76) and/or the V_(H) domain of antibody2C1 (e.g., SEQ ID NO:68).

The present invention further provides an antibody or antigen-bindingfragment thereof that binds ILT4 and comprises the light chainimmunoglobulin of antibody 1E1 (e.g., SEQ ID NO:3, 4, 5, 6, 7 or 45)and/or the heavy chain immunoglobulin of antibody 1E1 (e.g., SEQ IDNO:1, 2, 44, 79, 80, or 81).

The present invention further provides an antibody or antigen-bindingfragment thereof that binds ILT4 and comprises the light chainimmunoglobulin of antibody 2A6 (e.g., SEQ ID NO:11) and/or the heavychain immunoglobulin of antibody 2A6 (e.g., SEQ ID NO:8, 9, 10, 82, 83,or 84).

The present invention further provides an antibody or antigen-bindingfragment thereof that binds ILT4 and comprises the light chainimmunoglobulin of antibody 3G7 (e.g., SEQ ID NO:13) and/or the heavychain immunoglobulin of antibody 3G7 (e.g., SEQ ID NO:12 or 85).

The present invention further provides an antibody or antigen-bindingfragment thereof that binds ILT4 and comprises the light chainimmunoglobulin of antibody 2C1 (e.g., SEQ ID NO:15) and/or the heavychain immunoglobulin of antibody 2C1 (e.g., SEQ ID NO:14 or 86).

The present invention further provides an antibody that consists of twoheavy chains and two light chains, wherein each light chain comprisesthe V_(L) or light chain immunoglobulin of antibody 1E1, 2A6, 3G7, or2C1, and each heavy chain comprises the V_(H) or heavy chainimmunoglobulin of antibody 1E1, 2A6, 3G7, or 2C1.

In one embodiment, the antibody consists of two heavy chains and twolight chains, wherein each light chain comprises the amino acid sequenceset forth in SEQ ID NO:58 and each heavy chain comprises the amino acidsequence set forth in SEQ ID NO:57.

In another embodiment, the antibody consists of two heavy chains and twolight chains, wherein each light chain comprises the amino acid sequenceset forth in SEQ ID NO:58 and each heavy chain comprises the amino acidsequence set forth in SEQ ID NO:57, wherein the light chain furthercomprises the amino acid sequence set forth in SEQ ID NO:90.

In yet another embodiment, the antibody consists of two heavy chains andtwo light chains, wherein each light chain comprises the amino acidsequence set forth in SEQ ID NO:58 and each heavy chain comprises theamino acid sequence set forth in SEQ ID NO:57, wherein the heavy chainfurther comprises the amino acid sequence set forth in SEQ ID NO:89.

In still another embodiment, the antibody consists of two heavy chainsand two light chains, wherein each light chain comprises the amino acidsequence set forth in SEQ ID NO:58 and each heavy chain comprises theamino acid sequence set forth in SEQ ID NO:57, wherein the light chainfurther comprises the amino acid sequence set forth in SEQ ID NO:90 andthe heavy chain further comprises the amino acid sequence set forth inSEQ ID NO:89.

In one embodiment, the antibody consists of two heavy chains and twolight chains, wherein each light chain comprises the amino acid sequenceset forth in SEQ ID NO:7 and each heavy chain comprises the amino acidsequence set forth in SEQ ID NO:2.

In another embodiment, the antibody consists of two heavy chains and twolight chains, wherein each light chain consists of the amino acidsequence set forth in SEQ ID NO:7 and each heavy chain consists of theamino acid sequence set forth in SEQ ID NO:2.

In an embodiment of the invention, the antibody or antigen-bindingfragment of the present invention comprises a V_(L) (with or withoutsignal sequence), e.g., the V_(L) in any of SEQ ID NO:58 or 70-77,having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative ornon-conservative amino acid substitutions; and/or a V_(H) (with orwithout signal sequence), e.g., the V_(H) in any of SEQ ID NO:57 or63-69, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservativeor non-conservative amino acid substitutions, while still binding toILT4.

The present invention also includes polypeptides comprising the aminoacid sequences disclosed herein, e.g. SEQ ID NOs: 1-39, 44, 45, 47-58,63-77, or 79-86, as well as polypeptides comprising such amino acidsequences with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or moreconservative or non-conservative amino acid substitutions therein.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises a light chain immunoglobulin, a heavy chain immunoglobulin, orboth a light and heavy chain immunoglobulin, wherein the light chainimmunoglobulin has at least 90% amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 11, 13, 15, or45, and/or the heavy chain immunoglobulin has at least 90% amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:1,2, 8, 9, 10, 12, 14, 44, 79, 80, 81, 82, 83, 84, 85, or 86.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises a light chain immunoglobulin, a heavy chain immunoglobulin, orboth a light and heavy chain immunoglobulin, wherein the light chainimmunoglobulin comprises a light chain variable domain having at least90% amino acid sequence identity to the amino acid sequence set forth inSEQ ID NO:70, 71, 72, 73, 58, 74, 75, 76, or 77, and/or the heavy chainimmunoglobulin comprises a heavy chain variable domain having at least90% amino acid sequence identity to the amino acid sequence set forth inSEQ ID NO:63, 57, 64, 65, 66, 67, 68, or 69.

In an embodiment of the invention, an immunoglobulin heavy chain of ananti-ILT4 antibody or antigen-binding fragment of the present inventionis operably linked to a signal sequence, e.g., comprising the amino acidsequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO:41) and/or an immunoglobulinlight chain of an anti-ILT4 antibody or antigen-binding fragment of thepresent invention is operably linked to a signal sequence, e.g.,comprising the amino acid sequence MSVPTQVLGLLLLWLTDARC (SEQ ID NO:42).

In an embodiment of the invention, an N-terminal glutamine (Q) of animmunoglobulin chain set forth herein (e.g., heavy and/or light) isreplaced with a pyroglutamic acid. In one embodiment, an N-terminal Q ofa heavy chain immunoglobulin is replaced with a pyroglutamic acid. Inanother embodiment, an N-terminal Q of a light chain immunoglobulin isreplaced with a pyroglutamic acid. In yet another embodiment, anN-terminal Q of a heavy chain immunoglobulin and an N-terminal Q of aheavy chain immunoglobulin are replaced with a pyroglutamic acid.

Further provided herein are antibodies or antigen-binding fragments thatbind to the same epitope of ILT4 (e.g., human ILT4) as any anti-ILT4antibody or antigen-binding fragment thereof disclosed herein (e.g.,1E1, 2A6, 3G7 or 2C1). In one embodiment, the epitope is LYREKKSASW (SEQID NO:59). In another embodiment, the epitope is TRIRPEL (SEQ ID NO:60).In yet another embodiment, the epitope is NGQF (SEQ ID NO:61). In stillanother embodiment, the epitope is HTGRYGCQ (SEQ ID NO:62). In certainembodiments, the antibody or antigen-binding fragment thereof binds tothe same epitope of human ILT4 as an antibody or antigen-bindingfragment thereof comprising the heavy chain and light chain amino acidsequences set forth in SEQ ID NOs:1 and 3; 2 and 4; 2 and 5; 2 and 6; 2and 7; 2 and 3; 8 and 11; 9 and 11; 10 and 11; 12 and 13; 14 and 15; 79and 3; 80 and 4; 80 and 5; 80 and 6; 80 and 7; 80 and 3; 82 and 11; 83and 11; 84 and 11; 85 and 13; and 86 and 15; respectively. In someembodiments, the antibody or antigen-binding fragment thereof binds tothe same epitope of human ILT4 as an antibody or antigen-bindingfragment thereof comprising the heavy chain variable domain and lightchain variable domain amino acid sequences set forth in SEQ ID NOs:63and 70; 57 and 71; 57 and 72; 57 and 73; 57 and 58; 57 and 70; 64 and74; 65 and 74; 66 and 74; 67 and 75; 68 and 76; respectively.

The present invention include antibodies and antigen-binding fragmentsthat cross-block the binding of any anti-ILT4 antibody orantigen-binding fragment thereof disclosed herein (e.g., 1E1, 2A6, 3G7or 2C1) to ILT4 (e.g., human ILT4) or compete with any anti-ILT4antibody or antigen-binding fragment thereof disclosed herein (e.g.,1E1, 2A6, 3G7 or 2C1) to ILT4 (e.g., human ILT4). The cross-blockingantibodies and antigen-binding fragments thereof discussed herein can beidentified based on their ability to block any of the antibodies orfragments specifically set forth herein from binding to ILT4, in bindingassays (e.g., bio-layer interferometry (BLI; for example FORTEBIO OCTETbinding assay; Pall ForteBio Corp; Menlo Park, Calif.), surface plasmonresonance (SPR), BIACore, ELISA, flow cytometry). For example, in anembodiment of the invention, when using BLI, the tip of a fiber-opticprobe is coated with ligand (e.g., ILT4) and acts as the biosensorwherein binding of anti-ILT4 antibody or antigen-binding fragment to theILT4 alters the interference pattern of white light reflected from theprobe layer bound to ILT4 and an internal reference layer. The shift isindicative of ILT4/anti-ILT4 binding. In an embodiment of the invention,the ILT4 coated tip is immersed in a solution of analyte containingantibody or antigen-binding fragment, e.g., in the well of either a 96-or 384-well plate. In an embodiment of the invention, the plate isshaken during reading to create orbital flow. To read the assay, whitelight is directed down the length of the fiber. As mentioned above,interference between light reflecting off the reference layer andimmobilized surfaces containing ILT4 of the tip creates a distinctivepattern of light returning up the fiber. As molecules bind to theimmobilized sensor surface, that pattern changes in proportion to theextent of binding. For example, assays can be used in which a ILT4(e.g., human ILT4) protein is immobilized on a BLI probe or plate, areference anti-ILT4 antibody or fragment binds to ILT4 (e.g., atsaturating concentration) and a test anti-ILT4 antibody or fragment isadded. The ability of the test antibody to compete with the referenceantibody for ILT4 binding is then determined. In the BLI format, lightinterference of the ILT4 complex is monitored to determine if the testantibody effectively competes with the reference antibody, e.g.,nanometers of light wavelength shift over time is monitored wherein ashift indicates additional binding of the test antibody and a lack ofcross-blocking. In an embodiment of the invention, in the BLI format,cross-blocking is qualitatively deemed to have occurred between theantibodies if no additional binding of test antibody is observed. In anembodiment of the invention, as a control, cross-blocking of thereference antibody with itself is confirmed; wherein the assay isdetermined to be operating correctly if the reference antibody cancross-block itself from ILT4 binding. The ability of a test antibody toinhibit the binding of the anti-ILT4 antibody or fragment 1E1, 2A6, 3G7or 2C1, to ILT4 (e.g., human ILT4) demonstrates that the test antibodycan cross-block the antibody or fragment for binding to ILT4 (e.g.,human ILT4) and thus, may, in some cases, bind to the same epitope onILT4 (e.g., human ILT4) as 1E1, 2A6, 3G7 and/or 2C1. As stated above,antibodies and fragments that bind to the same epitope as any of theanti-ILT4 antibodies or fragments of the present invention also formpart of the present invention. In an embodiment of the invention, BLI isconducted in a sandwich format wherein a reference anti-ILT4 antibody orantigen-binding fragment is immobilized to the probe and then bound withILT4. Test anti-ILT4 antibody or antigen-binding fragment is then testedfor the ability to block binding of the references antibody or fragment.

In certain embodiments, the antibody or antigen-binding fragment thereofcompetes for binding to human ILT4 with an antibody or fragmentcomprising the heavy chain and light chain amino acid sequences setforth in SEQ ID NOs:1 and 3; 2 and 4; 2 and 5; 2 and 6; 2 and 7; 2 and3; 8 and 11; 9 and 11; 10 and 11; 12 and 13; 14 and 15; 79 and 3; 80 and4; 80 and 5; 80 and 6; 80 and 7; 80 and 3; 82 and 11; 83 and 11; 84 and11; 85 and 13; and 86 and 15; respectively. In some embodiments, theantibody or antigen-binding fragment thereof competes for binding tohuman ILT4 with an antibody or fragment comprising the heavy chainvariable domain and light chain variable domain amino acid sequences setforth in SEQ ID NOs:63 and 70; 57 and 71; 57 and 72; 57 and 73; 57 and58; 57 and 70; 64 and 74; 65 and 74; 66 and 74; 67 and 75; 68 and 76;respectively.

The present invention includes anti-ILT4 antibodies and antigen-bindingfragments thereof comprising N-linked glycans that are typically addedto immunoglobulins produced in Chinese hamster ovary cells (CHO N-linkedglycans) or to engineered yeast cells (engineered yeast N-linkedglycans), such as, for example, Pichia pastoris. For example, in anembodiment of the invention, the anti-ILT4 antibodies andantigen-binding fragments thereof comprise one or more of the“engineered yeast N-linked glycans” or “CHO N-linked glycans” that areset forth in FIG. 16 (e.g., G0 and/or G0-F and/or G1 and/or G1-F and/orand/or G2-F and/or Man5). In an embodiment of the invention, theanti-ILT4 antibodies and antigen-binding fragments thereof comprise theengineered yeast N-linked glycans, i.e., G0 and/or G1 and/or G2,optionally, further including Man5. In an embodiment of the invention,the anti-ILT4 antibodies and antigen-binding fragments thereof comprisethe CHO N-linked glycans, G0-F, G1-F and G2-F, optionally, furtherincluding G0 and/or G1 and/or G2 and/or Man5. In an embodiment of theinvention, about 80% to about 95% (e.g., about 80-90%, about 85%, about90% or about 95%) of all N-linked glycans on the anti-ILT4 antibodiesand antigen-binding fragments thereof are engineered yeast N-linkedglycans or CHO N-linked glycans. See Nett et al. Yeast. 28(3): 237-252(2011); Hamilton et al. Science. 313(5792): 1441-1443 (2006); Hamiltonet al. Curr Opin Biotechnol. 18(5): 387-392 (2007). For example, in anembodiment of the invention, an engineered yeast cell is GFI5.0 orYGLY8316 or strains set forth in U.S. Pat. No. 7,795,002 or Zha et al.Methods Mol Biol. 988:31-43 (2013). See also international patentapplication publication no. WO2013/066765.

Polynucleotides

The present invention comprises polynucleotides (e.g., DNA or RNA)encoding the immunoglobulin chains of anti-ILT4 antibodies andantigen-binding fragments thereof disclosed herein. For example, thepresent invention includes the nucleic acids encoding immunoglobulinheavy and/or light chains of antibodies 1E1, 2A6, 3G7 and 2C1 (e.g., SEQID NOs: 1-15, 44 and 45 or a variable domain thereof) as describedherein as well as nucleic acids which hybridize thereto.

The present invention includes polynucleotides encoding an antibodylight chain variable (V_(L)) domain comprising a CDR-L1, CDR-L2 andCDR-L3 of 1E1 (e.g., comprising the amino acids set forth in SEQ ID NOs:19-21); and/or an antibody heavy chain variable (V_(H)) domaincomprising a CDR-H1, CDR-H2 and CDR-H3 of 1E1 (e.g., comprising theamino acids set forth in SEQ ID NOs: 16-18).

The present invention includes polynucleotides encoding an antibodylight chain variable (V_(L)) domain comprising a CDR-L1, CDR-L2 andCDR-L3 of 2A6 (e.g., comprising the amino acids set forth in SEQ ID NOs:25-27); and/or an antibody heavy chain variable (V_(H)) domaincomprising a CDR-H1, CDR-H2 and CDR-H3 of 2A6 (e.g., comprising theamino acids set forth in SEQ ID NOs: 22-24).

The present invention includes polynucleotides encoding an antibodylight chain variable (V_(L)) domain comprising a CDR-L1, CDR-L2 andCDR-L3 of 3G7 (e.g., comprising the amino acids set forth in SEQ ID NOs:31-33); and/or an antibody heavy chain variable (V_(H)) domaincomprising a CDR-H1, CDR-H2 and CDR-H3 of 3G7 (e.g., comprising theamino acids set forth in SEQ ID NOs: 28-30).

The present invention includes polynucleotides encoding an antibodylight chain variable (V_(L)) domain comprising a CDR-L1, CDR-L2 andCDR-L3 of 2C1 (e.g., comprising the amino acids set forth in SEQ ID NOs:37-39); and/or an antibody heavy chain variable (V_(H)) domaincomprising a CDR-H1, CDR-H2 and CDR-H3 of 2C1 (e.g., comprising theamino acids set forth in SEQ ID NOs: 34-36).

The present invention includes polynucleotides encoding the V_(L) domainof antibody 1E1 (e.g., SEQ ID NO:70, 71, 72, 73, 58 or 77) and/or theV_(H) domain of antibody 1E1 (e.g., SEQ ID NO:63, 57 or 69).

The present invention includes polynucleotides encoding the V_(L) domainof antibody 2A6 (e.g., SEQ ID NO:74) and/or the V_(H) domain of antibody2A6 (e.g., SEQ ID NO:64, 65 or 66).

The present invention includes polynucleotides encoding the V_(L) domainof antibody 3G7 (e.g., SEQ ID NO:75) and/or the V_(H) domain of antibody3G7 (e.g., SEQ ID NO:67).

The present invention includes polynucleotides encoding the V_(L) domainof antibody 2C1 (e.g., SEQ ID NO:76) and/or the V_(H) domain of antibody2C1 (e.g., SEQ ID NO:68).

The present invention includes polynucleotides encoding the light chainimmunoglobulin of antibody 1E1 (e.g., SEQ ID NO: 3, 4, 5, 6, 7 or 45)and/or the heavy chain immunoglobulin of antibody 1E1 (e.g., SEQ ID NO:1, 2 or 44).

The present invention includes polynucleotides encoding the light chainimmunoglobulin of antibody 2A6 (e.g., SEQ ID NO: 11) and/or the heavychain immunoglobulin of antibody 2A6 (e.g., SEQ ID NO: 8, 9 or 10).

The present invention includes polynucleotides encoding the light chainimmunoglobulin of antibody 3G7 (e.g., SEQ ID NO: 13) and/or the heavychain immunoglobulin of antibody 3G7 (e.g., SEQ ID NO: 12).

The present invention includes polynucleotides encoding the light chainimmunoglobulin of antibody 2C1 (e.g., SEQ ID NO: 15) and/or the heavychain immunoglobulin of antibody 2C1 (e.g., SEQ ID NO: 14).

In one specific embodiment, the polynucleotide comprises nucleotidesequence set forth in SEQ ID NO:87. In another specific embodiment, thepolynucleotide comprises nucleotide sequence set forth in SEQ ID NO:88.In yet another embodiment, the polynucleotide comprises nucleotidesequence set forth in SEQ ID NO:87 and nucleotide sequence set forth inSEQ ID NO:88.

This present invention also provides expression vectors comprising theisolated nucleic acids of the invention, wherein the nucleic acid isoperably linked to one or more control sequences, e.g., that arerecognized by a host cell when the host cell is transfected with thevector. Also provided are host cells comprising an expression vector ofthe present invention. In certain embodiments, the host cells are CHOcells.

Methods of Making Antibodies and Antigen-Binding Fragments Thereof

The anti-ILT4 antibodies and antigen-binding fragments disclosed herein(e.g., 1E1, 2A6, 3G7 and/or 2C1) may be produced recombinantly. In thisembodiment, nucleic acids encoding one or more of the immunoglobulinchains of the antibodies and antigen-binding fragments of the invention(e.g., any one of SEQ ID NOs:1-15, 44, 45, or 79-86, or comprising aV_(H) or V_(L) of 1E1, 2A6, 3G7 or 2C1 as set forth in any one of SEQ IDNos:57, 58, 63-77) may be inserted into a vector and/or into a host cellchromosome and expressed in a recombinant host cell. There are severalmethods by which to produce recombinant antibodies which are known inthe art.

Antibodies (e.g., 1E1, 2A6, 3G7 or 2C1) can be recovered from theculture medium using standard protein purification methods. Further,expression of immunoglobulin chains of the invention from productioncell lines can be enhanced using a number of known techniques. Forexample, the glutamine synthetase gene expression system (the GS system)is a common approach for enhancing expression under certain conditions.The present invention includes vectors comprising one or morepolynucleotides encoding one or more of said immunoglobulin chains and aglutamine synthetase (GS) gene. In an embodiment of the invention, thevector is in a host cell that lacks functional glutamine synthetase. Inan embodiment of the invention, the host cell is in a culture mediumsubstantially lacking glutamine. Methods for making one or more of suchimmunoglobulin chains or an anti-ILT4 antibody or antigen-bindingfragment thereof comprising culturing such a host cell in culture mediumsubstantially lacking glutamine are within the scope of the presentinvention as well as such chains, antibodies and fragments produced bysuch a method.

In general, glycoproteins produced in a particular cell line ortransgenic animal will have a glycosylation pattern that ischaracteristic for glycoproteins produced in the cell line or transgenicanimal. Therefore, the particular glycosylation pattern of animmunoglobulin chain or antibody or antigen-binding fragment containingan immunoglobulin chain will depend on the particular cell line ortransgenic animal used to produce the antibody. Antibodies andantigen-binding fragments with a glycosylation pattern comprising onlynon-fucosylated N-glycans may be advantageous, because these antibodiesand antigen-binding fragments have been shown to typically exhibit morepotent efficacy than their fucosylated counterparts both in vitro and invivo (See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473(2003); U.S. Pat. Nos. 6,946,292 and 7,214,775). These anti-ILT4antibodies and antigen-binding fragments (e.g., 1E1, 2A6, 3G7 or 2C1)with non-fucosylated N-glycans are part of the present invention.

The present invention further includes antibody fragments of theanti-ILT4 antibodies disclosed herein (e.g., 1E1, 2A6, 3G7 or 2C1). Theantibody fragments include F(ab)₂ fragments, which may be produced byenzymatic cleavage of an IgG by, for example, pepsin. Fab fragments maybe produced by, for example, reduction of F(ab)₂ with dithiothreitol ormercaptoethylamine. A Fab fragment is a V_(L)—CL chain appended to aV_(H)-CH1 chain by a disulfide bridge. A F(ab)₂ fragment is two Fabfragments which, in turn, are appended by two disulfide bridges. The Fabportion of an F(ab)₂ molecule includes a portion of the Fc regionbetween which disulfide bridges are located. An FV fragment is a V_(L)or V_(H) region.

The present invention includes anti-ILT4 antibodies and antigen-bindingfragments thereof (e.g., 1E1, 2A6, 3G7 or 2C1) which are of the IgA,IgD, IgE, IgG and IgM classification. In one embodiment, the anti-ILT4antibody or antigen-binding fragment comprises a V_(H) as set forthherein and a heavy chain constant region, e.g., a human constant region,such as γ1, γ2, γ3, or γ4 human heavy chain constant region or a variantthereof. In an embodiment of the invention, the antibody orantigen-binding fragment comprises a V_(H) as set forth herein and alight chain constant region, e.g., a human light chain constant region,such as lambda or kappa human light chain region or variant thereof. Byway of example, and not limitation the human heavy chain constant regioncan be γ1 or γ4 and the human light chain constant region can be kappa.By way of example, and not limitation the human heavy chain constantregion can be γ1 or γ4 and the human light chain constant region can belambda. In an embodiment of the invention, the Fc region of the antibodyis γ4 with a Ser228Pro mutation (Schuurman, J et al., Mol. Immunol. 38:1-8, 2001).

In some embodiments of the invention, different constant domains may beappended to V_(L) and V_(H) regions derived from the CDRs providedherein. For example, if a particular intended use of an antibody orantigen-binding fragment of the present invention were to call foraltered effector functions, a heavy chain constant domain other thanhuman IgG1 may be used, or hybrid IgG1/IgG4 may be utilized. Suchanti-ILT4 antibodies and antigen-binding fragments thereof (e.g., 1E1,2A6, 3G7 or 2C1) and hybrids thereof comprising IgG1 and IgG4 constantdomains are part of the present invention.

In one embodiment, the IgG4 constant domain differs from the nativehuman IgG4 constant domain (Swiss-Prot Accession No. P01861.1) at aposition corresponding to position 228 in the EU system and position 241in the KABAT system, where the native Sen 08 is replaced with Pro, inorder to prevent a potential inter-chain disulfide bond between Cys106and Cys109 (corresponding to positions Cys 226 and Cys 229 in the EUsystem and positions Cys 239 and Cys 242 in the KABAT system) that couldinterfere with proper intra-chain disulfide bond formation. See Angal etal. (1993) Mol. Imunol. 30:105. In other instances, a modified IgG1constant domain which has been modified to increase half-life or reduceeffector function can be used. Such anti-ILT4 antibodies andantigen-binding fragments thereof (e.g., 1E1, 2A6, 3G7 or 2C1),comprising a modified IgG4 constant domain are part of the presentinvention.

The present invention includes recombinant methods for making ananti-ILT4 antibody or antigen-binding fragment thereof of the presentinvention (e.g., 1E1, 2A6, 3G7 or 2C1, or an immunoglobulin chainthereof, comprising (i) introducing a polynucleotide encoding one ormore immunoglobulin chains of the antibody or fragment (e.g., comprisingan amino acid sequence that includes any one or more of the sequencesset forth in SEQ ID NOs: 1-15, 44 and/or 45), for example, wherein thepolynucleotide is in a vector and/or is operably linked to a promoter(e.g., a viral promoter, a CMV promoter or SV40 promoter); (ii)culturing the host cell (e.g., a mammalian host cell, a fungal hostcell, a bacterial host cell, a Chinese hamster ovary (CHO) cell, an NSOcell, an SP2 cell, a HeLa cell, a baby hamster kidney (BHK) cell, amonkey kidney cell (COS), a human hepatocellular carcinoma cell (e.g.,Hep G2), a A549 cell, a 3T3 cell, a HEK-293 cell, a Pichia cell or aPichia pastoris cell) under condition favorable to expression of thepolynucleotide(s) and, (iii) optionally, isolating the antibody orfragment or chain from the host cell and/or medium in which the hostcell is grown. When making an antibody or antigen-binding fragmentcomprising more than one immunoglobulin chain, e.g., an antibody thatcomprises two heavy immunoglobulin chains and two light immunoglobulinchains, co-expression of the chains in a single host cell leads toassociation of the chains, e.g., in the cell or on the cell surface oroutside the cell if such chains are secreted, so as to form the antibodyor antigen-binding fragment molecule. The methods of the presentinvention include those wherein only a heavy immunoglobulin chain oronly a light immunoglobulin chain (e.g., any of those discussed hereinincluding mature fragments and/or variable domains thereof) isexpressed. Such chains are useful, for example, as intermediates in theexpression of an antibody or antigen-binding fragment that includes sucha chain.

Antibody Engineering of the Fc Region

The anti-ILT4 antibodies and antigen-binding fragments of the presentinvention (e.g., 1E1, 2A6, 3G7 and/or 2C1) can also be engineered toinclude modifications within the Fc region, typically to alter one ormore functional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or effector function(e.g., antigen-dependent cellular cytotoxicity). Furthermore, theantibodies and fragments disclosed herein can be chemically modified(e.g., one or more chemical moieties can be attached to the antibody) orbe modified to alter its glycosylation, again to alter one or morefunctional properties of the antibody. Each of these embodiments isdescribed in further detail below. The numbering of residues in the Fcregion is that of the EU index of Kabat.

The antibodies and antigen-binding fragments disclosed herein (e.g.,1E1, 2A6, 3G7 and/or 2C1) also include antibodies with modified (orblocked) Fc regions to provide altered effector functions. See, e.g.,U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571; WO2006/0057702.Such modification can be used to enhance or suppress various reactionsof the immune system, with possible beneficial effects in diagnosis andtherapy. Alterations of the Fc region include amino acid changes(substitutions, deletions and/or insertions), glycosylation ordeglycosylation, and adding multiple Fc domains. Changes to the Fc canalso alter the half-life of antibodies in therapeutic antibodies,enabling less frequent dosing and thus increased convenience anddecreased use of material. See Presta (2005) J. Allergy Clin. Immunol.116:731 at 734-35.

In one embodiment of the invention, the anti-ILT4 antibody orantigen-binding fragment (e.g., 1E1, 2A6, 3G7 and/or 2C1) is an IgG4isotype antibody or fragment comprising a serine to proline mutation ata position corresponding to position 228 (5228P; EU index) in the hingeregion of the heavy chain constant region. This mutation has beenreported to abolish the heterogeneity of inter-heavy chain disulfidebridges in the hinge region (Angal et al. supra; position 241 is basedon the Kabat numbering system). In one embodiment of the invention, thehinge region of CH1 of an anti-ILT4 antibody or antigen-binding fragmentthereof of the present invention (e.g., 1E1, 2A6, 3G7 and/or 2C1) ismodified such that the number of cysteine residues in the hinge regionis increased or decreased (e.g., by ±1, 2 or 3). This approach isdescribed further in U.S. Pat. No. 5,677,425. The number of cysteineresidues in the hinge region of CH1 is altered, for example, tofacilitate assembly of the light and heavy chains or to increase ordecrease the stability of the antibody.

In another embodiment, the Fc hinge region of an anti-ILT4 antibody orantigen-binding fragment (e.g., 1E1, 2A6, 3G7 and/or 2C1) is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745.

In another embodiment, the anti-ILT4 antibody or antigen-bindingfragment (e.g., 1E1, 2A6, 3G7 and/or 2C1) is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375. Alternatively, toincrease the biological half-life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the anti-ILT4 antibody or antigen-bindingfragment (e.g., 1E1, 2A6, 3G7 and/or 2C1). For example, one or moreamino acids selected from amino acid residues 234, 235, 236, 237, 297,318, 320 and 322 can be replaced with a different amino acid residuesuch that the antibody has an altered affinity for an effector ligandbut retains the antigen-binding ability of the parent antibody. Theeffector ligand to which affinity is altered can be, for example, an Fcreceptor or the C1 component of complement. This approach is describedin further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the anti-ILT4 antibody or antigen-binding fragment(e.g., 1E1, 2A6, 3G7 and/or 2C1) has altered C1 q binding and/or reducedor abolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351.

In yet another example, the Fc region is modified to increase ordecrease the ability of the anti-ILT4 antibody or antigen-bindingfragment (e.g., 1E1, 2A6, 3G7 and/or 2C1) to mediate antibody dependentcellular cytotoxicity (ADCC) and/or to increase or decrease the affinityof the antibody or fragment for an Fcγ receptor by modifying one or moreamino acids at the following positions: 238, 239, 243, 248, 249, 252,254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280,283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305,307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334,335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416,419, 430, 434, 435, 437, 438 or 439. This approach is described furtherin PCT Publication WO 00/42072. Moreover, the binding sites on humanIgG1 for FcγR1, FcγRI I, FcγRIII and FcRn have been mapped and variantswith improved binding have been described (see Shields et al. (2001) J.Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290,298, 333, 334 and 339 were shown to improve binding to FcγRIII.Additionally, the following combination mutants were shown to improveFcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A andS298A/E333A/K334A.

In one embodiment, the Fc region is modified to decrease the ability ofthe anti-ILT4 antibody or antigen-binding fragment (e.g., 1E1, 2A6, 3G7and/or 2C1) to mediate effector function and/or to increaseanti-inflammatory properties by modifying residues 243 and 264. In oneembodiment, the Fc region of the antibody is modified by changing theresidues at positions 243 and 264 to alanine. In one embodiment, the Fcregion is modified to decrease the ability of the antibody to mediateeffector function and/or to increase anti-inflammatory properties bymodifying residues 243, 264, 267 and 328.

Post-Translational Modifications

In still another embodiment, the antibody comprises a particularglycosylation pattern. For example, an aglycosylated anti-ILT4 antibodyor antigen-binding fragment thereof (e.g., 1E1, 2A6, 3G7 and/or 2C1),which lacks glycosylation, is part of the present invention. Theglycosylation pattern of an antibody or fragment may be altered to, forexample, increase the affinity or avidity of the antibody for anantigen. Such modifications can be accomplished by, for example,altering one or more of the glycosylation sites within the antibody orfragment sequence. For example, one or more amino acid substitutions canbe made that result removal of one or more of the variable regionframework glycosylation sites to thereby eliminate glycosylation at thatsite. Such aglycosylation may increase the affinity or avidity of theantibody or fragment for antigen. See, e.g., U.S. Pat. Nos. 5,714,350and 6,350,861.

An anti-ILT4 antibody or antigen-binding fragment (e.g., 1E1, 2A6, 3G7and/or 2C1) of the invention may also be made in which the glycosylationpattern includes hypofucosylated or afucosylated glycans, such ashypofucosylated antibodies and antigen-binding fragments or afucosylatedantibodies and fragments that have reduced amounts of fucosyl residueson the glycan. The antibody or antigen-binding fragment may also includeglycans having an increased amount of bisecting GlcNac structures. Suchaltered glycosylation patterns have been demonstrated to increase theADCC ability of antibodies. Such modifications can be accomplished by,for example, expressing the antibody or fragment in a host cell in whichthe glycosylation pathway was been genetically engineered to produceglycoproteins with particular glycosylation patterns. These cells havebeen described in the art and can be used as host cells in which toexpress recombinant antibodies of the invention to thereby produce anantibody with altered glycosylation. For example, the cell lines Ms704,Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (a(1,6)-fucosyltransferase), such that antibodies expressed in the Ms704,Ms705, and Ms709 cell lines lack fucose on their carbohydrates. Thepresent invention includes anti-ILT4 antibodies and antigen-bindingfragments lacking fucose or produced by a host cell that lacks FUT8. TheMs704, Ms705, and Ms709 FUT8^(−/−) cell lines were created by thetargeted disruption of the FUT8 gene in CHO/DG44 cells using tworeplacement vectors (see U.S. Patent Publication No. 20040110704 andYamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As anotherexample, EP 1176195 describes a cell line with a functionally disruptedFUT8 gene, which encodes a fucosyl transferase, such that antibodiesexpressed in such a cell line exhibit hypofucosylation by reducing oreliminating the α-1,6 bond-related enzyme. EP 1,176,195 also describescell lines which have a low enzyme activity for adding fucose to theN-acetylglucosamine that binds to the Fc region of the antibody or doesnot have the enzyme activity, for example the rat myeloma cell lineYB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 describes a variantCHO cell line, Lec13 cells, with reduced ability to attach fucose toAsn(297)-linked carbohydrates, also resulting in hypofucosylation ofantibodies expressed in that host cell (see also Shields et al. (2002)J. Biol. Chem. 277:26733-26740). Antibodies with a modifiedglycosylation profile can also be produced in chicken eggs, as describedin PCT Publication WO 06/089231. Alternatively, antibodies with amodified glycosylation profile can be produced in plant cells, such asLemna (U.S. Pat. No. 7,632,983). Methods for production of antibodies ina plant system are disclosed in the U.S. Pat. Nos. 6,998,267 and7,388,081. PCT Publication WO 99/54342 describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).Anti-ILT4 antibodies an antigen-binding fragments thereof of the presentinvention which are produced by such host cells are part of the presentinvention.

Alternatively, the fucose residues of the anti-ILT4 antibody orantigen-binding fragment (e.g., 1E1, 2A6, 3G7 and/or 2C1) can be cleavedoff using a fucosidase enzyme; e.g., the fucosidase α-L-fucosidaseremoves fucosyl residues from antibodies (Tarentino et al. (1975)Biochem. 14:5516-23). The present invention includes antibodies andfragments which have been treated with a fucosidase enzyme.

Anti-ILT4 antibody or antigen-binding fragments (e.g., 1E1, 2A6, 3G7and/or 2C1) disclosed herein further include those produced in lowereukaryote host cells, in particular fungal host cells such as yeast andfilamentous fungi have been genetically engineered to produceglycoproteins that have mammalian- or human-like glycosylation patterns(See for example, Choi et al, (2003) Proc. Natl. Acad. Sci. 100:5022-5027; Hamilton et al., (2003) Science 301: 1244-1246; Hamilton etal., (2006) Science 313: 1441-1443). A particular advantage of thesegenetically modified host cells over currently used mammalian cell linesis the ability to control the glycosylation profile of glycoproteinsthat are produced in the cells such that compositions of glycoproteinscan be produced wherein a particular N-glycan structure predominates(see, e.g., U.S. Pat. Nos. 7,029,872 and 7,449,308). These geneticallymodified host cells have been used to produce antibodies that havepredominantly particular N-glycan structures (See for example, Li etal., (2006) Nat. Biotechnol. 24: 210-215).

Antibody Conjugates

The anti-ILT4 antibodies and antigen-binding fragments disclosed herein(e.g., 1E1, 2A6, 3G7 and/or 2C1) may also be conjugated to a peptide orchemical moiety. The chemical moiety may be, inter alia, a polymer, aradionuclide or a therapeutic or cytotoxic agent. In particularembodiments of the invention, the chemical moiety is a polymer whichincreases the half-life of the antibody molecule in the body of asubject. Polymers include, but are not limited to, hydrophilic polymerswhich include but are not limited to polyethylene glycol (PEG) (e.g.,PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30kDa or 40 kDa), dextran and monomethoxypolyethylene glycol (mPEG). Lee,et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEG conjugatedsingle-chain antibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553)disclose conjugating antibodies with PEG which is attached to aradiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).

The antibodies and antigen-binding fragments disclosed herein (e.g.,1E1, 2A6, 3G7 and/or 201) may also be conjugated with labels, such asradiolabels. Labels include but are not limited to ⁹⁹Tc, ⁹⁰Y, ¹¹¹In,³²P, ¹⁴C, ¹²⁵I, ³H, ¹³¹I, ¹¹C, ¹⁵O, ¹³N, ¹⁸F, ³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra,⁶⁰Co, ⁵⁹Fe, ⁵⁷Se, ¹⁵²Eu, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th,and ⁴⁰K, ¹⁵⁷Gd, ⁵⁵Mn, ⁵²Tr, and ⁵⁶Fe.

The anti-ILT4 antibodies and antibody fragments disclosed herein (e.g.,1E1, 2A6, 3G7 and/or 2C1) may also be PEGylated (e.g., with 1 PEG or a 3kDa, 12 kDa or 40 kDa PEG polymer molecule), for example to increase itsbiological (e.g., serum) half-life. To PEGylate an antibody orantigen-binding fragment thereof, the antibody or fragment typically isreacted with a reactive form of polyethylene glycol (PEG), such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody orantigen-binding fragment. In particular embodiments, the PEGylation iscarried out via an acylation reaction or an alkylation reaction with areactive PEG molecule (or an analogous reactive water-soluble polymer).As used herein, the term “polyethylene glycol” is intended to encompassany of the forms of PEG that have been used to derivatize otherproteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycolor polyethylene glycol-maleimide. In certain embodiments, the antibodyto be PEGylated is an aglycosylated antibody. Methods for PEGylatingproteins are known in the art and can be applied to the antibodies ofthe invention. See, e.g., EP 0 154 316 and EP 0 401 384.

The anti-ILT4 antibodies and antigen-binding fragments disclosed herein(e.g., 1E1, 2A6, 3G7 and/or 2C1) may also be conjugated with labels suchas fluorescent or chemiluminescent labels, including fluorophores suchas rare earth chelates, fluorescein and its derivatives, rhodamine andits derivatives, isothiocyanate, phycoerythrin, phycocyanin,allophycocyanin, o-phthaladehyde, fluorescamine, ¹⁵²Eu, dansyl,umbelliferone, luciferin, luminal label, isoluminal label, an aromaticacridinium ester label, an imidazole label, an acridimium salt label, anoxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones,biotin/avidin, spin labels and stable free radicals.

The anti-ILT4 antibodies and antigen-binding fragments disclosed herein(e.g., 1E1, 2A6, 3G7 and/or 2C1) may also be conjugated to a cytotoxicfactor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain,ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins and compounds (e.g., fatty acids), dianthin proteins,Phytoiacca americana proteins PAPI, PAPII, and PAP-S, Momordicacharantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor,mitogellin, restrictocin, phenomycin, and enomycin.

Any method known in the art for conjugating the antibodies andantigen-binding to the various moieties may be employed, including thosemethods described by Hunter, et al., (1962) Nature 144:945; David, etal., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth.40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methodsfor conjugating antibodies are conventional and very well known in theart.

Pharmaceutical Compositions and Administration

The pharmaceutical compositions comprising the anti-ILT4 antibodies(e.g., fully human antibodies such as antagonist fully human antibodies(e.g., 1E1, 2A6, 3G7 and/or 2C1) and antigen-binding fragments thereofcan be prepared for storage by mixing the antibodies or antigen-bindingfragments thereof having the desired degree of purity with optionallyphysiologically acceptable carriers, excipients, or stabilizers (see,e.g., Remington, Remington's Pharmaceutical Sciences (18th ed. 1980)) inthe form of aqueous solutions or lyophilized or other dried forms.

In one embodiment, the pharmaceutical composition comprises an antibodythat consists of two heavy chains and two light chains, wherein eachlight chain consists of the amino acid sequence set forth in SEQ ID NO:7and each heavy chain consists of the amino acid sequence set forth inSEQ ID NO:2.

In another embodiment, the pharmaceutical composition comprises: (i) anantibody that consists of two heavy chains and two light chains, whereineach light chain consists of the amino acid sequence set forth in SEQ IDNO:7 and each heavy chain consists of the amino acid sequence set forthin SEQ ID NO:2, and (ii) pembrolizumab.

Formulations of therapeutic and diagnostic agents may be prepared bymixing with acceptable carriers, excipients, or stabilizers in the formof, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

Toxicity and therapeutic efficacy of the anti-ILT4 antibody orantigen-binding fragment (e.g., 1E1, 2A6, 3G7 and/or 2C1) compositions,administered alone or in combination with another therapeutic agent, canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose effective in 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index (LD₅₀/ED₅₀). In particular aspects, antibodiesexhibiting high therapeutic indices are desirable. The data obtainedfrom these cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration.

In a further embodiment, a further therapeutic agent that isadministered to a subject in association with an anti-ILT4 antibody(e.g., fully human antibody such as antagonist fully human antibodies)or antigen-binding fragment thereof disclosed herein (e.g., 1E1, 2A6,3G7 and/or 2C1) is administered to the subject in accordance with thePhysicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (Nov.1, 2002)).

The mode of administration can vary. Routes of administration includeoral, rectal, transmucosal, intestinal, parenteral; intramuscular,subcutaneous, intradermal, intramedullary, intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, intraocular,inhalation, insufflation, topical, cutaneous, transdermal, orintra-arterial.

The present invention provided methods for administering an anti-ILT4antibody or antigen-binding fragment thereof (e.g., 1E1, 2A6, 3G7 and/or2C1) comprising introducing the antibody or fragment into the body of asubject. For example, the method comprises piercing the body of thesubject with a needle of a syringe and injecting the antibody orfragment into the body of the subject, e.g., into the vein, artery,tumor, muscular tissue or subcutis of the subject.

The present invention provides a vessel (e.g., a plastic or glass vial,e.g., with a cap or a chromatography column, hollow bore needle or asyringe cylinder) comprising any of the anti-ILT4 antibodies orantigen-binding fragments (e.g., 1E1, 2A6, 3G7 and/or 2C1) set forthherein or a pharmaceutical composition thereof comprising apharmaceutically acceptable carrier.

The present invention also provides an injection device comprising anyof the anti-ILT4 antibodies or antigen-binding fragments (e.g., 1E1,2A6, 3G7 and/or 2C1) set forth herein or a pharmaceutical compositionthereof. An injection device is a device that introduces a substanceinto the body of a subject via a parenteral route, e.g., intramuscular,subcutaneous or intravenous. For example, an injection device may be asyringe (e.g., pre-filled with the pharmaceutical composition, such asan auto-injector) which, for example, includes a cylinder or barrel forholding fluid to be injected (e.g., comprising the antibody or fragmentor a pharmaceutical composition thereof), a needle for piecing skinand/or blood vessels for injection of the fluid; and a plunger forpushing the fluid out of the cylinder and through the needle bore. In anembodiment of the invention, an injection device that comprises anantibody or antigen-binding fragment thereof of the present invention ora pharmaceutical composition thereof is an intravenous (IV) injectiondevice. Such a device includes the antibody or fragment or apharmaceutical composition thereof in a cannula or trocar/needle whichmay be attached to a tube which may be attached to a bag or reservoirfor holding fluid (e.g., saline; or lactated ringer solution comprisingNaCl, sodium lactate, KCl, CaCl₂) and optionally including glucose)introduced into the body of the subject through the cannula ortrocar/needle.

The pharmaceutical compositions disclosed herein may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Suchneedleless devices comprising the pharmaceutical composition are alsopart of the present invention. The pharmaceutical compositions disclosedherein may also be administered by infusion. Examples of well-knownimplants and modules for administering the pharmaceutical compositionsinclude those disclosed in: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments. Many other such implants, delivery systems, and modulesare well known to those skilled in the art and those comprising thepharmaceutical compositions of the present invention are within thescope of the present invention.

Antibodies (e.g., fully human antibodies such as antagonist fully humanantibodies) or antigen-binding fragments thereof disclosed herein (e.g.,1E1, 2A6, 3G7 and/or 2C1) may be provided by continuous infusion, or bydoses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly,monthly, bimonthly, quarterly, semiannually, annually etc. Doses may beprovided, e.g., intravenously, subcutaneously, topically, orally,nasally, rectally, intramuscular, intracerebrally, intraspinally, or byinhalation. An effective dose of an anti-ILT4 antibody orantigen-binding fragment thereof of the present invention, is from about0.05 mg/kg (body weight) to about 30 mg/kg (body weight), e.g., fortreatment or prevention of cancer or infectious diseases.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment. Generally, in determining the dose, the dose begins with anamount somewhat less than the optimum dose and it is increased by smallincrements thereafter until the desired or optimum effect is achievedrelative to any negative side effects. Important diagnostic measuresinclude those of symptoms of, e.g., the inflammation or level ofinflammatory cytokines produced. In general, it is desirable that abiologic that will be used is derived from the same species as theanimal targeted for treatment, thereby minimizing any immune response tothe reagent. In the case of human subjects, for example, chimeric andfully human antibodies are may be desirable. Guidance in selectingappropriate doses of anti-ILT4 antibodies or fragments is available(see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub.Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies,Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.)(1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases,Marcel Dekker, New York, N.Y.; Baert et al. (2003) New Engl. J. Med.348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973;Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al.(2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J.Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602).

Whether a disease symptom has been alleviated can be assessed by anyclinical measurement typically used by physicians or other skilledhealthcare providers to assess the severity or progression status ofthat symptom. While an embodiment of the present invention (e.g., atreatment method or article of manufacture) may not be effective inalleviating the target disease symptom(s) in every subject, it shouldalleviate the target disease symptom(s) in a statistically significantnumber of subjects as determined by any statistical test known in theart such as the Student's t-test, the chi²-test, the U-test according toMann and Whitney, the Kruskal-Wallis test (H-test),Jonckheere-Terpstra-test and the Wilcoxon-test.

Therapeutic Uses of Anti-ILT4 Antibodies

The present invention also provides methods for treating or preventingcancer in subjects, such as human subjects, in need of such treatment byadministering an effective amount of the anti-ILT4 antibodies orantigen-binding fragments thereof of the present invention which aredisclosed herein (e.g., 1E1, 2A6, 3G7 and/or 2C1) which may be effectivefor such treatment or prevention.

In certain embodiments, the cancer is solid tumor. In other embodiments,the cancer is hematologic cancer. In certain embodiments, the cancer ismetastatic. In some embodiments, the cancer is relapsed. In otherembodiments, the cancer is refractory. In yet other embodiments, thecancer is relapsed and refractory.

In some embodiments, the cancer is anaplastic astrocytoma, astrocytoma,bladder cancer, bone cancer, brain cancer, breast cancer (e.g.,characterized by a mutation in BRCA1 and/or BRCA2), carcinoid cancer,cervical cancer, chondrosarcoma, choroid plexus papilloma, colorectalcancer, endometrial cancer, ependymoma, esophagus cancer, Ewing'ssarcoma, gall bladder cancer, gastric cancer, glioblastoma, head andneck cancer, hepatoblastoma, hepatocellular carcinoma, idiopathicmyelfibrosis, kidney cancer, leukemia, liver cancer, lung cancer (e.g.,non-small cell lung cancer), lymphoma, medulloblastoma, melanoma,meningioma, Merkel cell cancer, mesothelioma, multiple myeloma,neuroblastoma, oligodendroglioma, osteosarcoma, ovarian cancer,pancreatic cancer, polycythemia vera, primitive neuroectodermal tumor,prostate cancer, renal cell cancer, renal transitional cell cancer,retinoblastoma, rhabdoid tumor of the kidney, rhabdomyosarcoma, salivarygland cancer, sarcoma, small intestine cancer, soft tissue sarcoma,squamous cell carcinoma (e.g., cutaneous squamous cell carcinoma),synovial sarcoma, thrombocythemia, thyroid cancer, uterine cancer,vestibular schwannoma, or Wilm's tumor. In an embodiment of theinvention, the cancer is metastatic cancer, e.g., of the varietiesdescribed above.

In an embodiment of the invention, the cancer is a myeloid-rich tumor(e.g., mesothelioma, kidney cancer, lymphoma, sarcoma, melanoma, head &neck cancer, breast cancer, bladder cancer, gastric cancer, ovariancancer or thyroid cancer; see e.g., Burt et al. Cancer. 117 (22):5234-44(2011); Dannenmann et al. Oncoimmunology 2(3):e23562 (2013); Steidl etal. N. Engl. J. Med. 362:875-885 (2010); Fujiwara et al., Am. J. Pathol.179(3):1157-70 (2011); Bronkhorst et al. Invest. Ophthalmol. Vis. Sci.52(2):643-50 (2011); Balermpas et al. Br. J. Cancer 111(8):1509-18(2014); and Zhang et al. PLoS One 7(12):e50946 (2012)). Since ILT4 isexpressed primarily by myeloid cells and granulocytes, and myeloid cellinfiltration into tumors is generally associated with poor prognosis dueto the immunosuppressive effects of these cells that can antagonizeanti-tumor responses by T-cells, treatment with an anti-ILT4 antibody orantigen-binding fragment of the present invention will benefit subjectswith a high myeloid or immunosuppressive myeloid cell infiltration.

Thus, provided herein are methods of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising (a) the CDR-L1, CDR-L2, and CDR-L3 of a light chain variable(V_(L)) domain of an immunoglobulin chain that comprises the amino acidsequence set forth in SEQ ID NO: 3-7, 11, 13, 15, or 45; and/or (b) theCDR-H1, CDR-H2, and CDR-H3 of a heavy chain variable (V_(H)) domain ofan immunoglobulin chain that comprises the amino acid sequence set forthin SEQ ID NO: 1, 2, 8-10, 12, 14, 44, or 79-86.

In an embodiment of the invention, the method of treating a cancer in ahuman subject in need thereof, comprising administering to the humansubject an effective amount of the antibody or antigen-binding fragmentthereof comprising: (1) a V_(H) domain comprising: complementaritydetermining region-H1 (CDR-H1): GYYWS (SEQ ID NO: 16), complementaritydetermining region-H2 (CDR-H2): EINHXGSTNYNPSLKS wherein X is S or A(SEQ ID NO: 17); and complementarity determining region-H3 (CDR-H3):LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:complementarity determining region-L1 (CDR-L1): TGSSSNIGAGYDVH (SEQ IDNO: 19), complementarity determining region-L2 (CDR-L2): GX₁X₂NRPS,wherein X₁ is S or A and X₂ is N, Q, E or D (SEQ ID NO: 20); andcomplementarity determining region-L3 (CDR-L3): QSFDNSLSAYV (SEQ ID NO:21); (2) a V_(H) domain comprising: CDR-H1: SYAIS (SEQ ID NO: 22);CDR-H2: GIIPIFGTANYAQKFQG (SEQ ID NO: 23); and CDR-H3:YFX₁X₂SGWYKGGAFDI, wherein X₁ is D or S and X₂ is S or A (SEQ ID NO:24); and/or, a V_(L) domain comprising: CDR-L1: TLRSGINVDTYRIH (SEQ IDNO: 25); CDR-L2: YKSDSDKHQGS (SEQ ID NO: 26); and CDR-L3: AIWYSSTWV (SEQID NO: 27); (3) a V_(H) domain comprising: CDR-H1: SYAMH (SEQ ID NO:28); CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO: 29); and CDR-H3:VGEWIQLWSPFDY (SEQ ID NO: 30); and/or, a V_(L) domain comprising:CDR-L1: RASQGISSWLA (SEQ ID NO: 31); CDR-L2: AASSLQS (SEQ ID NO: 32);and CDR-L3: QQYNSYPPT (SEQ ID NO: 33); and/or (4) a V_(H) domaincomprising: CDR-H1: ELSMH (SEQ ID NO: 34); CDR-H2: GFDPEDGETIYAQKFQG(SEQ ID NO: 35); and CDR-H3: AGPLYTIFGVVIIPDNWFDP (SEQ ID NO: 36);and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:37); CDR-L2: GNSNRPS (SEQ ID NO: 38); and CDR-L3: QSYDSSLSGSGW (SEQ IDNO: 39).

In one embodiment, the method of treating a cancer in a human subject inneed thereof, comprising administering to the human subject an effectiveamount of the antibody or antigen-binding fragment thereof comprising:CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47),and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domaincomprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO:47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domaincomprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQID NO: 51), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GDSNRPS(SEQ ID NO: 52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the method of treating a cancer in a human subject inneed thereof, comprising administering to the human subject an effectiveamount of the antibody or antigen-binding fragment thereof comprising:CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47),and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domaincomprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQID NO: 53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GEANRPS(SEQ ID NO: 55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GDANRPS(SEQ ID NO: 56), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the method of treating a cancer in a human subject inneed thereof, comprising administering to the human subject an effectiveamount of the antibody or antigen-binding fragment thereof comprising:CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48),and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domaincomprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GESNRPS(SEQ ID NO: 51), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GDSNRPS(SEQ ID NO: 52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the method of treating a cancer in a human subject inneed thereof, comprising administering to the human subject an effectiveamount of the antibody or antigen-binding fragment thereof comprising:CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48),and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domaincomprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQID NO: 53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GEANRPS(SEQ ID NO: 55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L)domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2:GDANRPS(SEQ ID NO: 56), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still other embodiments, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: a light chain immunoglobulin, a heavy chain immunoglobulin,or both a light and heavy chain immunoglobulin, wherein the light chainimmunoglobulin comprises the amino acid sequence set forth in SEQ IDNO:3, 4, 5, 6, 7, 11, 13, 15, or 45; and/or the heavy chainimmunoglobulin comprises the amino acid sequence set forth in SEQ ID NO:1, 2, 8, 9, 10, 12, 14, 44, 79, 80, 81, 82, 83, 84, 85, or 86. In yetstill other embodiments, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: a light chain immunoglobulin, a heavy chain immunoglobulin,or both a light and heavy chain immunoglobulin, wherein the light chainvariable domain comprises the amino acid sequence set forth in SEQ IDNO:70, 71, 72, 73, 58, 74, 75, 76, or 77, and/or the heavy chainvariable domain comprise the amino acid sequence set forth in SEQ IDNO:63, 57, 64, 65, 66, 67, 68, or 69.

In more embodiments, the method of treating a cancer in a human subjectin need thereof, comprising administering to the human subject aneffective amount of the antibody or antigen-binding fragment thereofcomprising: any of the following sets of heavy chain immunoglobulins andlight chain immunoglobulins: (1) a heavy chain immunoglobulin comprisingthe amino acid sequence set forth in SEQ ID NO:1; a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:3; (2) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:2; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:4; (3) a heavychain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:2; a light chain immunoglobulin comprising the amino acid sequenceset forth in SEQ ID NO:5; (4) a heavy chain immunoglobulin comprisingthe amino acid sequence set forth in SEQ ID NO:2; a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:6; (5) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:2; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:7; (6) a heavychain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:2; a light chain immunoglobulin comprising the amino acid sequenceset forth in SEQ ID NO:3; (7) a heavy chain immunoglobulin comprisingthe amino acid sequence set forth in SEQ ID NO:8; a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:11; (8) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:9; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:11; (9) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:10; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:11; (10) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:12; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:13; (11) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:14; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:15; (12) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:79; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:3; (13) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:80; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:4; (14) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:80; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:5; (15) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:80; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:6; (16) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:80; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:7; (17) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:80; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:3; (18) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:82; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:11; (19) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:83; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:11; (20) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:84; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:11; (21) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:85; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:13; or (22) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:86; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:15.

In even more embodiments, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: any of the following sets of heavy chain variable domain andlight chain variable domain: (1) a heavy chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:63; and alight chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:70; (2) a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:57; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:71; (3)a heavy chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:57; and a light chain variable domain comprising theamino acid sequence set forth in SEQ ID NO:72; (4) a heavy chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO:57; and a light chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:73; (5) a heavy chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:57; and alight chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:58; (6) a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:57; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:70; (7)a heavy chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:64; and a light chain variable domain comprising theamino acid sequence set forth in SEQ ID NO:74; (8) a heavy chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO:65; and a light chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:74; (9) a heavy chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:66; and alight chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:74; (10) a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:67; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:75; or(11) a heavy chain variable domain comprising the amino acid sequenceset forth in SEQ ID NO:68; and a light chain variable domain comprisingthe amino acid sequence set forth in SEQ ID NO:76.

In one preferred embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: a V_(H) domain comprising the amino acid sequence set forthin SEQ ID NO:57; and a V_(L) domain comprising the amino acid sequenceset forth in SEQ ID NO:58.

In another preferred embodiment, the method of treating a cancer in ahuman subject in need thereof, comprising administering to the humansubject an effective amount of the antibody or antigen-binding fragmentthereof comprising: a heavy chain immunoglobulin comprising the aminoacid sequence set forth in SEQ ID NO:2; and a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:7. In yetanother preferred embodiment, the method of treating a cancer in a humansubject in need thereof, comprising administering to the human subjectan effective amount of the antibody or antigen-binding fragment thereofcomprising: a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:80; and a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:7.

Further provided herein are methods of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereofcomprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising (a) the CDR-L1,CDR-L2, and CDR-L3 of a light chain variable (V_(L)) domain of animmunoglobulin chain that comprises the amino acid sequence set forth inSEQ ID NO: 3-7, 11, 13, 15, or 45; and/or (b) the CDR-H1, CDR-H2, andCDR-H3 of a heavy chain variable (V_(H)) domain of an immunoglobulinchain that comprises the amino acid sequence set forth in SEQ ID NO: 1,2, 8-10, 12, 14, 44, or 79-86.

In an embodiment of the invention, the method of blocking binding ofILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in needthereof, comprising administering to the human subject an effectiveamount of the antibody or antigen-binding fragment thereof comprising:(1) a V_(H) domain comprising: complementarity determining region-H1(CDR-H1): GYYWS (SEQ ID NO: 16), complementarity determining region-H2(CDR-H2): EINHXGSTNYNPSLKS wherein X is S or A (SEQ ID NO: 17); andcomplementarity determining region-H3 (CDR-H3): LPTRWVTTRYFDL (SEQ IDNO: 18); and/or, a V_(L) domain comprising: complementarity determiningregion-L1 (CDR-L1): TGSSSNIGAGYDVH (SEQ ID NO: 19), complementaritydetermining region-L2 (CDR-L2): GX₁X₂NRPS, wherein X₁ is S or A and X₂is N, Q, E or D (SEQ ID NO: 20); and complementarity determiningregion-L3 (CDR-L3): QSFDNSLSAYV (SEQ ID NO: 21); (2) a V_(H) domaincomprising: CDR-H1: SYAIS (SEQ ID NO: 22); CDR-H2: GIIPIFGTANYAQKFQG(SEQ ID NO: 23); and CDR-H3: YFX₁X₂SGWYKGGAFDI, wherein X₁ is D or S andX₂ is S or A (SEQ ID NO: 24); and/or, a V_(L) domain comprising: CDR-L1:TLRSGINVDTYRIH (SEQ ID NO: 25); CDR-L2: YKSDSDKHQGS (SEQ ID NO: 26); andCDR-L3: AIWYSSTWV (SEQ ID NO: 27); (3) a V_(H) domain comprising:CDR-H1: SYAMH (SEQ ID NO: 28); CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO:29); and CDR-H3: VGEWIQLWSPFDY (SEQ ID NO: 30); and/or, a V_(L) domaincomprising: CDR-L1: RASQGISSWLA (SEQ ID NO: 31); CDR-L2: AASSLQS (SEQ IDNO: 32); and CDR-L3: QQYNSYPPT (SEQ ID NO: 33); and/or (4) a V_(H)domain comprising: CDR-H1: ELSMH (SEQ ID NO: 34); CDR-H2:GFDPEDGETIYAQKFQG (SEQ ID NO: 35); and CDR-H3: AGPLYTIFGVVIIPDNWFDP (SEQID NO: 36); and/or, a V_(L) domain comprising: CDR-L1: TGSSSNIGAGYDVH(SEQ ID NO: 37); CDR-L2: GNSNRPS (SEQ ID NO: 38); and CDR-L3:QSYDSSLSGSGW (SEQ ID NO: 39).

In one embodiment, the method of blocking binding of ILT4 to HLA-G,HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprisingadministering to the human subject an effective amount of the antibodyor antigen-binding fragment thereof comprising: CDR-H1: GYYWS (SEQ IDNO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of blocking binding of ILT4 to HLA-G,HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprisingadministering to the human subject an effective amount of the antibodyor antigen-binding fragment thereof comprising: CDR-H1: GYYWS (SEQ IDNO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: GYYWS (SEQ IDNO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: CDR-H1: GYYWS(SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the method of blocking binding of ILT4 to HLA-G,HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprisingadministering to the human subject an effective amount of the antibodyor antigen-binding fragment thereof comprising: CDR-H1: GYYWS (SEQ IDNO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of blocking binding of ILT4 to HLA-G,HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprisingadministering to the human subject an effective amount of the antibodyor antigen-binding fragment thereof comprising: CDR-H1: GYYWS (SEQ IDNO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: CDR-H1: GYYWS(SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: CDR-H1: GYYWS(SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the method of blocking binding of ILT4 to HLA-G,HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprisingadministering to the human subject an effective amount of the antibodyor antigen-binding fragment thereof comprising: CDR-H1: GYYWS (SEQ IDNO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of blocking binding of ILT4 to HLA-G,HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprisingadministering to the human subject an effective amount of the antibodyor antigen-binding fragment thereof comprising: CDR-H1: GYYWS (SEQ IDNO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: CDR-H1: GYYWS(SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: CDR-H1: GYYWS(SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the method of blocking binding of ILT4 to HLA-G,HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprisingadministering to the human subject an effective amount of the antibodyor antigen-binding fragment thereof comprising: CDR-H1: GYYWS (SEQ IDNO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of blocking binding of ILT4 to HLA-G,HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprisingadministering to the human subject an effective amount of the antibodyor antigen-binding fragment thereof comprising: CDR-H1: GYYWS (SEQ IDNO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: CDR-H1: GYYWS(SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: CDR-H1: GYYWS(SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3:LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V_(L) domain comprising:CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56),and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still other embodiments, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: a light chainimmunoglobulin, a heavy chain immunoglobulin, or both a light and heavychain immunoglobulin, wherein the light chain immunoglobulin comprisesthe amino acid sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 11, 13,15, or 45; and/or the heavy chain immunoglobulin comprises the aminoacid sequence set forth in SEQ ID NO: 1, 2, 8, 9, 10, 12, 14, 44, 79,80, 81, 82, 83, 84, 85, or 86.

In yet still other embodiments, the method of blocking binding of ILT4to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: a light chainimmunoglobulin, a heavy chain immunoglobulin, or both a light and heavychain immunoglobulin, wherein the light chain variable domain comprisesthe amino acid sequence set forth in SEQ ID NO:70, 71, 72, 73, 58, 74,75, 76, or 77, and/or the heavy chain variable domain comprise the aminoacid sequence set forth in SEQ ID NO:63, 57, 64, 65, 66, 67, 68, or 69.

In more embodiments, the method of blocking binding of ILT4 to HLA-G,HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprisingadministering to the human subject an effective amount of the antibodyor antigen-binding fragment thereof comprising: any of the followingsets of heavy chain immunoglobulins and light chain immunoglobulins: (1)a heavy chain immunoglobulin comprising the amino acid sequence setforth in SEQ ID NO:1; a light chain immunoglobulin comprising the aminoacid sequence set forth in SEQ ID NO:3; (2) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:2; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:4; (3) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:2; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:5; (4) a heavychain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:2; a light chain immunoglobulin comprising the amino acid sequenceset forth in SEQ ID NO:6; (5) a heavy chain immunoglobulin comprisingthe amino acid sequence set forth in SEQ ID NO:2; a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:7; (6) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:2; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:3; (7) a heavychain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:8; a light chain immunoglobulin comprising the amino acid sequenceset forth in SEQ ID NO:11, (8) a heavy chain immunoglobulin comprisingthe amino acid sequence set forth in SEQ ID NO:9; a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:11; (9) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:10; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:11; (10) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:12; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:13; (11) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:14; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:15; (12) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:79; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:3; (13) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:80; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:4; (14) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:80; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:5; (15) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:80; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:6; (16) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:80; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:7; (17) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:80; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:3; (18) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:82; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:11, (19) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:83; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:11; (20) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:84; a lightchain immunoglobulin comprising the amino acid sequence set forth in SEQID NO:11; (21) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:85; a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:13; or (22) aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:86; a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:15.

In even more embodiments, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: any of thefollowing sets of heavy chain variable domain and light chain variabledomain: (1) a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:63; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:70; (2) aheavy chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:57; and a light chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:71; (3) a heavy chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:57; anda light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:72; (4) a heavy chain variable domain comprising theamino acid sequence set forth in SEQ ID NO:57; and a light chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO:73; (5) a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:57; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:58; (6) aheavy chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:57; and a light chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:70; (7) a heavy chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:64; anda light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:74; (8) a heavy chain variable domain comprising theamino acid sequence set forth in SEQ ID NO:65; and a light chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO:74; (9) a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:66; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:74; (10) aheavy chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:67; and a light chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:75; or (11) a heavy chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:68; anda light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:76.

In one preferred embodiment, the method of blocking binding of ILT4 toHLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: a V_(H) domaincomprising the amino acid sequence set forth in SEQ ID NO:57; and aV_(L) domain comprising the amino acid sequence set forth in SEQ IDNO:58.

In another preferred embodiment, the method of blocking binding of ILT4to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof,comprising administering to the human subject an effective amount of theantibody or antigen-binding fragment thereof comprising: a heavy chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:2; and a light chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO:7.

In yet another preferred embodiment, the method of blocking binding ofILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in needthereof, comprising administering to the human subject an effectiveamount of the antibody or antigen-binding fragment thereof comprising: aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:80; and a light chain immunoglobulin comprising the aminoacid sequence set forth in SEQ ID NO:7.

The present invention also provides methods for treating or preventingan infectious disease in a subject by administering an effective amountof anti-ILT4 antibodies or antigen-binding fragments thereof disclosedherein (e.g., 1E1, 2A6, 3G7 and/or 2C1) to the subject which may beeffective for such treatment or prevention. In an embodiment of theinvention, the infectious disease is viral infection. In an embodimentof the invention, the infectious disease is bacterial infection. In anembodiment of the invention, the infectious disease is parasiticinfection. In an embodiment of the invention, the infectious disease isfungal infection.

The present invention includes methods of treating any of the cancers orinfectious diseases discussed herein by administering a therapeuticallyeffective amount of an anti-ILT4 antibody or antigen-binding fragmentthereof (e.g., 1E1, 2A6, 3G7 and/or 2C1) optionally in association withany of the further chemotherapeutic agents or therapeutic proceduresdiscussed herein as well as compositions including such an antibody orfragment in association with such a further chemotherapeutic agent(e.g., co-formulated antibody or fragment and further chemotherapeuticagent).

A “subject” is a mammal such as, for example, a human, dog, cat, horse,cow, mouse, rat, monkey (e.g., cynomolgous monkey, e.g., Macacafascicularis or Macaca mulatta) or rabbit.

In an embodiment of the invention, an anti-ILT4 antibody (e.g., fullyhuman antibody such as antagonist fully human antibodies) orantigen-binding fragment thereof of the present invention (e.g., 1E1,2A6, 3G7 and/or 2C1) is in association with a further chemotherapeuticagent such as an antibody or antigen-binding fragment thereof. In anembodiment of the invention, the further chemotherapeutic agent is anantiemetic, erythropoietin, GM-CSF, a vaccine, an anti-PD-L1 antibody orantigen-binding fragment thereof, an anti-PD-L2 antibody orantigen-binding fragment thereof, an anti-PD1 antibody orantigen-binding fragment thereof (e.g., pembrolizumab or nivolumab),5-fluorouracil (5-FU), a platinum compound, bevacizumab, daunorubicin,doxorubicin, temozolomide topotecan, irinotecan, paclitaxel, docetaxel,imatinib or rituximab.

The term “in association with” indicates that the components, ananti-ILT4 antibody (e.g., fully human antibody such as antagonist fullyhuman antibodies) or antigen-binding fragment thereof of the presentinvention (e.g., 1E1, 2A6, 3G7 and/or 2C1) along with another agent suchas pembrolizumab or nivolumab, can be formulated into a singlecomposition, e.g., for simultaneous delivery, or formulated separatelyinto two or more compositions (e.g., a kit). Each component can beadministered to a subject at a different time than when the othercomponent is administered; for example, each administration may be givennon-simultaneously (e.g., separately or sequentially) at intervals overa given period of time. Moreover, the separate components may beadministered to a subject by the same or by a different route (e.g.,wherein an anti-ILT4 antibody (e.g., fully human antibody such asantagonist fully human antibodies) or antigen-binding fragment thereof(e.g., 1E1, 2A6, 3G7 and/or 2C1 is administered parenterally andpaclitaxel is administered orally).

Assays and Experimental and Diagnostic Uses

The present invention includes any method for forming a complex betweenan anti-ILT4 antibody or antigen-binding fragment thereof of the presentinvention and ILT4 or a fragment thereof comprising contacting the ILT4polypeptide or fragment with the anti-ILT4 antibody or antigen-bindingfragment under conditions suitable for binding and complex formation.

The anti-ILT4 antibodies (e.g., fully human antibodies such asantagonist fully human antibodies) and antigen-binding fragments thereofdisclosed herein (e.g., 1E1, 2A6, 3G7 and/or 2C1) may be used asaffinity purification agents. In this process, the anti-ILT4 antibodiesand antigen-binding fragments thereof are immobilized on a solid phasesuch a sephadex, glass or agarose resin or filter paper, using methodswell known in the art. The immobilized antibody or fragment is contactedwith a sample containing the ILT4 protein (or a fragment thereof) to bepurified, and, thereafter, the support is washed with a suitable solventthat will remove the material in the sample except the ILT4 proteinwhich is bound to the immobilized antibody or fragment. Finally, thesupport is washed with a solvent which elutes the bound ILT4 (e.g.,protein A). Such immobilized antibodies and fragments form part of thepresent invention.

The present invention includes cell-based ELISA methods using theanti-ILT4 antibodies and antigen-binding fragments thereof of thepresent invention (e.g., 1E1, 2A6, 3G7 and/or 2C1). In an embodiment ofthe invention, the method is for determining whether cells contain ILT4and the method includes the steps: (i) contacting said cells immobilizedto a solid surface (e.g., a microplate), which are to be tested for thepresence of ILT4, with an anti-ILT4 antibody or antigen-binding fragmentthereof of the present invention, (ii) optionally, washing the mixtureto remove unbound anti-ILT4 antibody or fragment, (iii) contacting theanti-ILT4 antibody or fragment with a labeled secondary antibody orantigen-binding fragment thereof that binds to the anti-ILT4 antibody orfragment, (iv) optionally washing the complex to remove unboundantibodies or fragments and (v) detecting the presence of the label onthe secondary antibody or fragment; wherein detection of the labelindicates that cells containing ILT4 are immobilized to the solidsurface.

The present invention includes ELISA assays (enzyme-linked immunosorbentassay) incorporating the use of an anti-ILT4 antibody (e.g., fully humanantibodies such as antagonist fully human antibodies) or antigen-bindingfragment thereof disclosed herein (e.g., 1E1, 2A6, 3G7 and/or 2C1). Forexample, such a method, for determining if a sample contains ILT4 or afragment thereof, comprises the following steps:

(a) coating a substrate (e.g., surface of a microtiter plate well, e.g.,a plastic plate) with anti-ILT4 antibody (e.g., fully human antibodiessuch as antagonist fully human antibodies) or antigen-binding fragmentthereof (e.g., 1E1, 2A6, 3G7 and/or 2C1);(b) applying a sample to be tested for the presence of ILT4 to thesubstrate;(c) washing the substrate, so that unbound material in the sample isremoved;(d) applying detectably labeled antibodies (e.g., enzyme-linkedantibodies) which are also specific to the ILT4 antigen;(e) washing the substrate, so that the unbound, labeled antibodies areremoved;(f) detecting the label on the antibodies; if the labeled antibodies areenzyme linked, apply a chemical which is converted by the enzyme into adetectable, e.g., fluorescent, signal; and

Detection of the label, e.g., associated with the substrate, indicatesthe presence of the ILT4 protein.

In an embodiment of the invention, the labeled antibody orantigen-binding fragment thereof is labeled with peroxidase which reactswith ABTS (e.g., 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid))or 3,3′,5,5′-Tetramethylbenzidine to produce a color change which isdetectable. Alternatively, the labeled antibody or fragment is labeledwith a detectable radioisotope (e.g., ³H) which can be detected byscintillation counter in the presence of a scintillant.

An anti-ILT4 antibody (e.g., fully human antibodies such as antagonistfully human antibodies) or antigen-binding fragment thereof of theinvention (e.g., 1E1, 2A6, 3G7 and/or 2C1) may be used in a Western blotor immune-protein blot procedure. Such a procedure forms part of thepresent invention and includes e.g.:

(1) providing a membrane or other solid substrate comprising a sample tobe tested for the presence of ILT4 or fragment thereof, e.g., optionallyincluding the step of transferring proteins from a sample to be testedfor the presence of ILT4 (e.g., from a PAGE or SDS-PAGE electrophoreticseparation of the proteins in the sample) onto a membrane or other solidsubstrate (e.g., using methods known in the art (e.g., semi-dry blottingor tank blotting)); and contacting the membrane or other solid substrateto be tested for the presence of bound ILT4 or a fragment thereof withan anti-ILT4 antibody or antigen-binding fragment thereof of theinvention (e.g., 1E1, 2A6, 3G7 and/or 2C1);(2) washing the membrane one or more times to remove unbound anti-ILT4antibody or fragment and other unbound substances; and(3) detecting the bound anti-ILT4 antibody or fragment.

Such a membrane may take the form, for example, of a nitrocellulose orvinyl-based (e.g., polyvinylidene fluoride (PVDF)) membrane to which theproteins to be tested for the presence of ILT4 in a non-denaturing PAGE(polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) gel have been transferred(e.g., following electrophoretic separation in the gel). Beforecontacting the membrane with the anti-ILT4 antibody or fragment, themembrane is optionally blocked, e.g., with non-fat dry milk or the likeso as to bind non-specific protein binding sites on the membrane.

Detection of the bound anti-ILT4 antibody or fragment indicates that theILT4 protein is present on the membrane or substrate and in the sample.Detection of the bound antibody or fragment may be by binding theantibody or fragment with a secondary antibody (an anti-immunoglobulinantibody) which is detectably labeled and, then, detecting the presenceof the secondary antibody label.

The anti-ILT4 antibodies (e.g., fully human antibodies such asantagonist fully human antibodies) and antigen-binding fragments thereofdisclosed herein (e.g., 1E1, 2A6, 3G7 and/or 2C1) may also be used forimmunohistochemistry. Such a method forms part of the present inventionand comprises, e.g.,

(1) contacting cells (e.g., myeloid lineage cells such as monocytes,macrophages or dendtritic cells) to be tested for the presence of ILT4protein with an anti-ILT4 antibody or antigen-binding fragment thereofof the invention (e.g., 1E1, 2A6, 3G7 and/or 2C1), and(2) detecting the antibody or fragment on or in the cells.

If the antibody or fragment itself is detectably labeled, it can bedetected directly. Alternatively, the antibody or fragment may be boundby a detectably labeled secondary antibody wherein the label is thendetected.

EXAMPLES

These examples are intended to exemplify the present invention and arenot a limitation thereof. Compositions and methods set forth in theExamples form part of the present invention.

As used herein the term “p1E1 (C1)” refers to a fully human anti-ILT41E1 mAb which is derived from the germline V genes, IGHV4-34*0 andIGLV1-40*01, respectively; having the human IgG1 and human lambdaconstant domains.

>heavy chain (SEQ ID NO: 44)QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPG K >light chain(SEQ ID NO: 45) QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECS

As used herein “p1E1(G4)” refers to a fully human anti-ILT4 1E1 (havingthe mutations Q1E in heavy chain and Q1E in light chain) mAb which isderived from the germline V genes, IGHV4-34*0 and IGLV1-40*01,respectively; having the human IgG4 (S228P) and human lambda constantdomains.

> heavy chain (SEQ ID NO: 1)EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK > human IgG4 (S228P) constant domain(SEQ ID NO: 89) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGK > light chain (SEQ ID NO: 3)ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVEGGGTQLTVLGQPKAAPSVTLEPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS > human lambda constant domain (SEQ ID NO: 90)GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS

As used herein “1E1(G4)” refers to a fully human anti-ILT4 1E1 (havingthe mutations Q1E and N53D in light chain and Q1E and S54A in heavychain) mAb which is derived from the germline V genes, IGHV4-34*0 andIGLV1-40*01, respectively; having human IgG4 (S228P) and human lambdaconstant domains.

> heavy chain (SEQ ID NO: 2)EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHAGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK > light chain(SEQ ID NO: 7) ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECS

Example 1: Anti-ILT4 Antibody Identification and CharacterizationAntibody Generation and Identification

The anti-ILT4 parental human mAb 1E1 was identified using the RETROCYTEDISPLAY platform (Breous-Nystroma et al., Methods 65(1): 57-67 (2014)).The RETROCYTE DISPLAY platform utilizes retroviral gene transfer ofhuman antibody genes into mammalian pre-B cells to generate stable highdiversity antibody display libraries. Human cord blood containing naïveB cells were used as the source material for antibody heavy and lightchains. The cellular antibody libraries typically expressed >10⁸different full length (hIgG1-4 isotypes) monoclonal human antibodies onthe cell surface of the pre-B cell.

Antibody Pre-panels were compiled from antibody hits identified in 3separate RETROCYTE DISPLAY screening campaigns. Candidates were enrichedbased on FACS detection of recombinant human ILT4 antigen-binding toB-cell clones. Putative B-cell clones were sorted out and their antibodysequences determined. These sequences were then used to produce antibodycandidates and ILT4 binding was confirmed using ILT4 CHO transfectantcell binding assays, with parental CHO cells serving as a negativecontrol. Candidates were also counter-screened for ILT4 specificityagainst closely related ILT family members (human ILT2, LILRA1, andLILRA2 CHO transfectant FACS) and against ILT family members by flowcytometric evaluation (LILRA1, LILRA2, LILRA4, LILRA5, LILRA6, ILT2,ILT5, and ILT3).

Monoclonal antibodies were further tested for their ability to bindcynomolgous monkey ILT4 (predicted sequence from NCBI) CHO transfectantsvia FACS. Candidate mAbs were also screened for their ability to blockrecombinant HLA-G Fc ligand binding to recombinant ILT4 protein inLuminex-based assays or to ILT4-expressing CHO cells in FACS-basedassays. Candidate antibody functional activity was assessed by threemethods: 1) Rescue of spontaneous I L2 suppression in ILT4-transfectedmouse 3A9 T-cells; 2) Rescue of HLA-G-dependent suppression ofCD200RLa-stimulated mast cell degranulation in mouse WTMC ILT4transfectants; and 3) Cytokine modulation in whole PBMC mixed lymphocytereactions.

Based on the above screening criteria, eight anti-ILT4 antibodies (1E1,1G2, 2A6, 2D5, 3E6, 3G7, 2C1 and 5A6) were selected based on theirfunctional and biophysical properties. Antibodies were further analyzedand re-evaluated in a set of bio-functional, biophysical, andphysicochemical assays. Functional assays assessed antibody-mediateddose-dependent rescue of I L-2 suppression of 3A9 cells and mast celldegranulation described above. Luminex and cell-based ligand blockingand ligand competition assays were performed and binding properties andaffinities to the ILT4 target antigen, off-target antigens, and PBMCsubsets were determined using Biacore and flow cytometry based assays,respectively. Biophysical assays assessed antibody stability(temperature, pH) and degradation and aggregation behavior. Sequenceliabilities and potential post-translational modification motifs wereaddressed in order to exclude potential antibody production risks.Finally, candidates were tested in an in vivo, tumor regression studyusing SKMEL5 melanoma-challenged humanized mice.

Biophysical Properties

Studies were conducted on human 1E1 sequence (human IgG4 backbone withS228P mutation) transiently expressed in CHO cells. 1E1 presented thefollowing physicochemical characteristics: calculated and experimentallydetermined isoelectric point (PI) were respectively ˜7.29 and ˜7.2,aggregation level (HMW species) was <5%, Tm onset >60° C., Tm1˜65.2° C.,Tm2˜78.8° C., and was stable for at least 5 freeze/thaw cycles. Thesequence of 1E1 originally had a N-glycosylation site in V_(H)-CDR2which was successfully corrected with the 554A mutation without negativeimpact on binding by BIACORE and in functional assay. Stress studiesshowed deamidation of the N53 residue in V_(L)-CDR3 (>13% under high PHconditions). N53 was successfully corrected (N53D) without a negativeimpact on binding and in a functional assay (rescue of IL-2 release fromILT4 3A9 T cell transfectants with 1E1). Both N-terminal Q residues inthe 1E1 V_(H) and V_(L) were mutated to E (V_(H)-Q1E and V_(L)-Q1E) toreduce risk of heterogeneity due to deamidation. Stress studiesconducted on the mutated 1E1 sequence (1E1 V_(H) Q1E, S54A/V_(L) Q1E,N53D) IgG4 S228P/Lambda) showed ˜17% oxidation of the W102 residue inV_(H)-CDR3 under forced oxidation with AAPH(2,2′-Azobis(2-amidinopropane) dihydrochloride) at 6 hours with minimalimpact on binding and function. AAPH is used to force oxidation of Trpand Met residues. Under the same condition, ˜8% oxidation of W7 in V_(H)framework was detected with no impact on binding and function. Around 8%afucosylated antibody was detected by peptide mapping. This findingmight be associated with the molecule being expressed in transientlytransfected CHO cells.

Epitope Mapping

The epitope on ILT4 of the antibody clone, p1E1(G1), was identified byHydrogen-Deuterium Exchange Mass Spectrometry. The antibody p1E1(G1) waspremixed with the recombinant histidine tagged, extracellular domain ofILT4, then the complex was incubated in deuterium buffer. The amount ofdeuterium incorporation was measured by mass spectrometry. ILT4 residuesLYREKKSASW (39-48 of ILT4 without signal sequence) (SEQ ID NO:59) andTRIRPEL (50-56 of ILT4 without signal sequence) (SEQ ID NO:60) and toless extent NGQF (59-62 of ILT4 without signal sequence) (SEQ ID NO:61)and HTGRYGCQ (71-78 of ILT4 without signal sequence) (SEQ ID NO:62) wereidentified as showing the largest difference in deuterium labelingcompared to an antigen-only sample, indicating they are likely theresidues interacting with p1E1(G1) (FIG. 1). These peptides are ondomain 1 of ILT4. Other peptides on domain 1 and 2 that showed lessdeuterium labeling differences are likely protected due toconformational stability upon antibody binding. No significantdifferences in labeling were seen in domains 3 and 4.

When mapped onto the crystal structure of human ILT4, the residuesprotected by p1E1(G4) are forming non-linear conformational epitopecomprising three beta-strands and a loop (FIG. 2A).

ILT4 uses two binding interfaces to engage its ligand HLA-G (Shiroishiet al, 2006): site 1 for beta-2-microglobulin binding, located in domain2 of ILT4, and site 2 for HLA-G heavy chain binding, located in domain 1of ILT4 (FIG. 2B). Site 1 includes ILT4 residues Trp-67, Asp-177,Asn-179, and Val-183 (numbering according to Shiroishi et al, 2006).Site 2 includes ILT4 residues Arg-36, Tyr-38, Lys-42, Ile-47, and Thr-48(numbering according to Shiroishi et al, 2006). The HDX-MS data of thisapplication show that Tyr-38, Lys-42, and Thr-48 (numbering according toShiroishi et al, 2006) are part of the p1E1(G1) epitope on ILT4 domain 1as residues Tyr-40, Lys-44, and Thr-50 of human ILT4 (FIG. 2A). Thisindicates that the human ILT4 epitope bound by p1E1(G1) overlaps withthe site 2 epitope bound by the HLA-G ligand.

Example 2: Affinity, Binding and Blocking Properties of Anti-ILT4 mAb1E1 Binding Affinities of 1E1(G4) and HLA-G1 to Human ILT4 Determined bySurface Plasmon Resonance (SPR)

Binding of 1E1(G4) and HLA-G1 Fc (recombinant extracellular domain ofHLA-G (isoform 1) fused to the human IgG1 Fc domain to make solubleHLA-G1 protein) to ILT4-His (recombinant extracellular domain of humanILT4 fused to a poly-Histidine tag to make soluble ILT4 protein) wasassessed via Biacore. Either 1E1(G4) or HLA-G1 Fc was captured on aBiacore chip via Fc capture. Monomeric ILT4-His was then tested forbinding and data indicated ILT4-His bound 1E1(G4) with a greater than600-fold higher affinity than HLA-G1 Fc (Table 3).

TABLE 3 ILT4-His binds 1E1(G4) with a greater than 600-fold tighteraffinity than HLA-G1 Fc. 1:1 binding kinetics and steady- state analysesindicate 630- and 670-fold differences. n Ligand ka (1/M⁻¹s⁻¹) kd (1/s)K_(D) (M) K_(D) Ratio 2 1E1 (G4) 5.5E+05 9.0E−03 1.7E−08 1 2 HLA-G1 Fc1.1E+05 1.1E+00 1.1E−05 630 (Kinetics) HLA-G1 Fc — — 1.1E−05 670 (SSA)

A surface plasmon resonance (SPR) assay on a Biacore T200 (GEHEALTHCARE) instrument was used to determine the monovalent affinitiesof anti-human ILT4 IgG4 mAb (1E1(G4)) and HLA-G1 Fc fusion (HLA-G1-Fc)against polyhistidine-tagged human ILT4 (ILT4-His). Either mAb or Fcfusion protein was captured on a CM5 sensor chip prepared using a HumanFc Capture kit (GE HEATHCARE) and a titrating concentration series ofILT4-His was injected over this surface. Biacore T200 EvaluationSoftware was used to fit each titration series to a 1:1 binding model.The association (ka, M⁻¹ s⁻¹) and dissociation (kd, s⁻¹) rate constantswere determined for each set of titrations and used to calculate thedissociation constant, K_(D) (M)=koff/kon, for each titration. As shownin Table 1, the monovalent affinities (K_(D)) of 1E1(G4) mAb andHLA-G1-Fc against human ILT4-His were 17 nM and 11 uM, respectively,with a 630-fold difference indicated by the K_(D) ratio. Because of thefast ka and kd kinetics constants, steady-state approximation (SSA) wasalso used to confirm the low affinity of HLA-G1-Fc for ILT4-His.

Blocking of HLA-G Binding to ILT4 3A9 T Cell Transfectants with 1E1(G4)

ILT4 3A9 T cell transfectants were pre-treated with 1E1(G4) or isotypecontrol at various doses, followed by secondary detection of 1E1(G4)(FIG. 3A) or by treatment with a fixed concentration of biotinylatedHLA-G1 Fc chimera to assess the ability of 1E1(G4) to block cognateligand (FIG. 3B). 1E1(G4) and HLA-G Fc was detected via flow cytometry.The data show 1E1(G4) blocked HLA-G1 Fc binding in a dose-dependentmanner.

Luminex- and Cell-Based Ligand Blocking and Competition Assays

Antibodies 1E1, 2A6, 2C1, and 3G7 were tested in Luminex- and cell-basedligand blocking and ligand competition assays for their potential ofinhibiting the interaction of recombinant dimeric HLA-G with ILT4antigen coupled to beads or expressed by CHO/ILT4+ cells.

The Luminex based ligand blocking and competition assays usedrecombinant human ILT4 antigen chemically coupled to Luminex beads. Inthe blocking assay, the beads were pre-incubated with a dose range(0.5-9,000 ng/mL in 1:3 serial dilutions) of hIgG4 variants of theanti-ILT4 antibody 1E1, 2A6, 2C1, or 3G7. Bead-bound ILT4 antigen wasthen tested for binding to soluble biotinylated HLA-G/Fc fusion proteinat a concentration of 50 nM. In the Luminex ligand competition assay,antigen-coupled Luminex beads were pre-incubated with solublebiotinylated HLA-G/Fc fusion protein at a concentration of 50 nM beforedose-titrations (0.5-9,000 ng/mL in 1:3 serial dilutions) of hIgG4variants of the anti-ILT4 antibody 1E1, 2A6, 2C1, or 3G7. In both assaysetups, ILT4:HLA-G interaction was detected with an anti-streptavidin-PEantibody and IC₅₀ values were determined. All tested antibodies showeddose-dependent blocking of HLA-G binding to Luminex bead-coupled ILT4/Fcand IC₅₀ values are summarized in Table 4. All tested antibodies alsoshowed dose-dependent competition with HLA-G for binding to Luminexbead-coupled ILT4/Fc (data not shown).

TABLE 4 Luminex Ligand Blocking Assay Antibody IC50 (ng/mL) 1E1 19.2 2A617.7 2C1 10.2 3G7 40.0

The cell-based ligand blocking and ligand competition assays followed asimilar principle as described for the Luminex-based assays and used aCHO/ILT4 cell line for surface expression of the antigen. In theblocking assay, cells were pre-incubated with the tested antibody usinga dose range of 1-20,000 ng/mL in 1:3 serial dilutions. ILT4 antigenswere then tested for binding to soluble biotinylated HLA-G/Fc fusionprotein at a concentration of 5 pg/ml. The competition assay used areverse setup. Cells were pre-incubated with soluble biotinylatedHLA-G/Fc fusion protein at a concentration of 5 pg/ml before dosetitrations of the tested antibody were added in a range of 1-20,000ng/mL in 1:3 serial dilutions. ILT4:HLA-G interaction was detected withan anti-streptavidin-PE antibody. All tested antibodies showeddose-dependent blocking of HLA-G binding to CHO-expressed ILT4 and IC₅ ₀values are summarized in Table 5. All tested antibodies also showeddose-dependent competition with HLA-G for binding to CHO-expressed ILT4(data not shown).

TABLE 5 Cell-based Ligand Blocking Assay Antibody IC50 (ng/mL) 1E1 98.42A6 232.6 2C1 56.1 3G7 374.3Blockade of Non-HLA-G MHC Class I Ligand Binding to ILT4 3A9 T CellTransfectants with p1E1(G1)

ILT4 3A9 T cell transfectants were pre-treated with p1E1(G1) or hIgG1isotype control at various doses, followed by treatment with a fixedconcentration of fluorochrome labeled HLA-F or CD1d tetramers, orHLA-A02:01 or HLA*B7:02 dexamers to assess the ability of p1E1(G1) toblock non-HLA-G MHC class I ligands. HLA-A, HLA-B, and HLA-F binding toILT4 was inhibited by p1E1(G1) in a dose titratable fashion (FIG. 4),indicating the ability of p1E1(G1) to block other reported MHC class Iligands.

Blockade of ANGPTL Binding to ILT4 3A9 T Cell Transfectants withp1E1(G1)

Angiopoietin-like (ANGPTL) proteins were recently reported to bind toILT4 expressed by human hematopoietic stem cells (Zheng et al., Nature.2012 May 30; 485(7400):656-60 and Deng et al. Blood. 2014 Aug. 7;124(6):924-35). To test whether p1E1(G1) could block ANGPTL familymember binding to ILT4, commercially available ANGPTL proteins orprotein fragments were purchased and tested for binding to ILT4 3A9 Tcell transfectants that were pre-treated with p1E1(G1). Binding dataindicate that ANGPTL1, 4, and potentially 7 could bind to ILT4 and notvector control cells at the concentration of protein tested. p1E1(G1)was able to fully block ANGPTL protein binding at a saturating dose(FIG. 5).

ILT Family Specificity Binding of 1E1(G4) to Human ILT 3A9 T CellTransfectants

ILT family specificity binding of 1E1(G4) to human ILT family memberswas assessed by cell-based flow cytometry using 3A9 T cell linestransfected to express human ILT4, ILT2, ILT3 (two variants), ILT5,LILRB5, LILRA1, LILRA2, ILT7, ILT8, or ILT11. 1E1(G4) specifically boundhuman ILT4 and did not have cross-reactivity to any other ILT familymember tested (FIGS. 6A and 6B).

Example 3: Bioactivity of Anti-ILT4 mAb 1E1 in Engineered and PrimaryCells Ability of 1E1(G4), 2A6, 2C1, and 3G7 to Reverse Interleukin 2Suppression in Engineered ILT4 3A9 Cell-Based Assays

An anti-CD3 antibody was used to stimulate control mouse 3A9 T-cellsresulting in an increase of IL-2 release. In contrast, ILT4 3A9 T-celltransfectants could not express IL-2 in the presence of CD3 stimulation,possibly due to cross-reactivity with ILT4 with mouse MHC class Imolecules or through an unknown xeno-ligand(s). This interactionappeared to lead to spontaneous multimerization/activation of the ILT4receptor, resulting in suppression of the anti-CD3 mediated IL-2release. Accordingly, antibodies that functionally bind to ILT4 andblock the interaction of ILT4 with the xeno-ligand(s) and/or inhibitreceptor multimerization should restore IL-release.

1E1(G4), 2A6, 2C1, and 3G7 were tested for mediating IL-2 release ofILT4 mouse 3A9 T-cell transfectants. 1E1(G4), 2A6, 2C1, or 3G7 was addedto ILT4+ mouse 3A9 T-cell transfectants and IL-2 release was measuredphotometrically by ELISA following 24 hours of anti-CD3 mediated cellstimulation. The representative dose response curve of 1E1(G4) is shownin FIG. 7. EC₅₀ values of the tested antibodies were determined from thedose response curves and shown in Table 6.

TABLE 6 IL-2 Repression Assay Antibody EC50 (μg/mL) 1E1 0.43 2A6 1.2 2C10.24 3G7 0.26

WTMC (Wild-Type Mast Cell) Mouse Cell Degranulation Assay (QualitativeOnly)

p1E1(G4) and 1E1(G4) were tested for rescue of ILT4:HLA-G dependent mastcell degranulation. Mouse WTMC mast cells were transfected with humanILT4 and stimulated with plate-bound antibody raised against CD200RLa.Antibody-mediated cross-linking of CD200RLa led to mast celldegranulation by activation of ITAM motifs found in the intracellulardomain of CD220RLa. Degranulation can be measured colorimetrically byassaying granule content release in assay supernatants. In the presenceof plate-bound HLA-G tetramer, CD200RLa-mediated mast cell degranulationwas inhibited in ILT4 transfectants. Pretreatment of ILT4 WTMCtransfectants with p1E1(G4) or 1E1(G4) before stimulation withplatebound anti-CD200RLa and HLA-G tetramer reversed degranulationinhibition in a dose titratable manner (FIG. 8).

Effect of 1E1(G4) on Myeloid Derived Cytokine Secretion by Primary HumanPeripheral Blood Mononuclear Cells (PBMC) Assessment of 1E1(G4) in HumanPrimary PBMC LPS Stimulation Assays

Expression of TNFa, a prototypical myeloid derived proinflammatorycytokine, was reported to be expressed by monocytes expressing lowlevels of ILT4 when stimulated with LPS. Monocytes with high expressionof ILT4 did not express as much TNFa, and the lack of ILT4 expressionwas found to be a hallmark of monocytes isolated from patients withpsoriatic arthritis (Bergamini et al., PLoS One. 2014 Mar. 27;9(3):e92018). ILT4 expression on monocytes could inhibit myeloid celleffector activity and antagonize proinflammatory cytokine induction(e.g., TNFα) in the presence of proinflammatory stimuli (e.g., LPS). Assuch, whole PBMCs isolated from healthy human donors were treated withLPS and the ability of ILT4 antagonism to enhance proinflammatorymyeloid cytokine expression was evaluated with 1E1(G4). FIGS. 9A and 9Bshow data from one of three experiments, with 3 donors each,demonstrating that 1E1(G4) enhanced LPS-dependent expression of bothGM-CSF and TNFa (both myeloid-derived cytokines) in a dose titratablefashion.

Assessment of 1E1(G4) in Human Primary PBMC Anti-CD3 Stimulation Assays

To assess whether 1E1(G4) treatment could enhance T-cell or myeloideffector cytokine expression in the presence of a sub-optimal T cellstimulus, whole PBMCs isolated from healthy human donors were treatedwith anti-CD3 to induce T-cell proliferation, and the ability ofILT4-antagonism to modify cytokine expression was evaluated with1E1(G4). FIGS. 10A and 10B show data from one of three experiments, with3 donors each, demonstrating that 1E1(G4) enhanced anti-CD3 dependentexpression of GM-CSF and TNFa in a dose titratable fashion.

Example 4: Anti-Tumor Efficacy of Anti-ILT4 Antibodies in the HumanizedMouse SK-MEL-5 Tumor Model Anti-Tumor Efficacy of 1E1, 2A6, 2C1, and 2D5in the Humanized Mouse Tumor Model

Antibodies 1E1, 2A6, 2C1 and 2D5 were tested in an in vivo tumorregression assay. Humanized mice (NSG mice reconstituted with humanhematopoeitic stem cells to establish human immune cell constitution)were inoculated with 1×10⁶ SKMEL5 melanoma cells (HLA class A*02:01) andtumor growth was monitored until an average size of 150 mm³ afterapproximately 35 days was observed. Seven randomized groups of mice,each containing six animals, were subcutaneously dosed with isotypecontrol antibody (hIgG1+hIgG4, 20 mg/kg of each), or 20 mg/kg of ofeither of the following anti-ILT4 antibodies: 1E1-IgG1, 1E1-IgG1 (N297Q)(Fc null mutant), 1E1-IgG4, 2A6-IgG4, 2C1-IgG4, and 2D5-IgG4. Mice weredosed every seven days (day 35, 42, and 49; total of three doses) andtumor size was measured until day 63. Results indicated impaired tumorgrowth in mice treated with 1E1 (IgG1, IgG1-(N297Q), IgG4), 2A6-IgG4,and 2D5-IgG4 compared to animals dosed with the isotype control (FIG.17). In contrast, mice treated with either isotype control or anti-ILT4candidate 2C1 failed to demonstrate impaired tumor growth.

Anti-Tumor Efficacy of p1E1(G4) in the Humanized Mouse Tumor Model

A humanized mouse tumor model was developed to test in vivo efficacy ofp1E1(G4) for tumor growth inhibition. Immuno-deficient NSG mice werereconstituted with human hematopoietic stem cells. After mice wereconfirmed to harbor peripheral human CD45+ immune cells (≥25% of PBMCs),they were inoculated with SK-MEL-5 tumor cells, a human skin melanomaderived tumor line. These cells were selected for their geneticexpression of HLA-G. Following inoculation, tumors were allowed to growto an approximate size of 150 mm³. Mice were randomized into groups andchallenged with either hIgG4 isotype control or p1E1(G4).

Mice treated with p1E1(G4) displayed tumor growth inhibition over thecourse of the study (FIG. 11A). One complete and one partial regressionwere observed with p1E1(G4) (FIG. 11C).

Anti-Tumor Efficacy of 1E1(G4) in the Humanized Mouse Tumor Model

The anti-tumor activity of 1E1(G4) was tested in the humanized mouseSK-MEL-5 tumor model. In this model, immunodeficient NSG(NOD.Cg-Prkdc^(scid)//2rg^(tm1Wji)/SzJ) mice are irradiated and injectedwith human CD34+ hematopoietic stem cells isolated from umbilical cordblood. After several months of engraftment, human immune cells can bedetected in mouse blood. The mice were then implanted subcutaneously(SC) with the human melanoma-derived SK-MEL-5 cell line.

For this study, NSG mice transplanted at 3 to 4 weeks of age with humancord blood-derived CD34+ cells from 3 separate donors were injected SCwith 1×10⁶ SK-MEL-5 cells at approximately 20 weeks of age. HumanizedNSG mice bearing SK-MEL-5 tumors were assigned to 2 treatment groups at6 mice per group (3 mice from each human CD34+ donor cohort pertreatment group) when mean tumor size was approximately 100 mm³, 21 daysfollowing tumor inoculation (DO tumor randomization). Tumor-bearing micewere injected SC with 20 mg/kg 1E1(G4) or a hIgG4 isotype control mAbevery 7 days for 4 doses. Tumor volumes were monitored every 7 daysfollowing the initiation of treatment. Anti-tumor efficacy in the1E1(G4) treatment group was significantly greater than the isotypecontrol group (p 0.001 from Day 28 through Day 49) (FIG. 12A). Theendpoint tumor weight in 1E1(G4)-treated mice was lower than that inisotype-treated mice (FIG. 12C). Overall, the results revealedsignificant anti-tumor efficacy of 1E1(G4) at 20 mg/kg in the humanizedmouse SK-MEL-5 tumor model. No effect was observed on body weight (FIG.12B) and splenic weight (FIG. 12D) with 1E1(G4) treatment.

Example 5: ILT4 Haplotype Binding of 1E1(G4) to Human ILT 3A9 T CellTransfectants

Single nucleotide polymorphism data from publicly available sources (1KGenome Project Phase 3) was used to determine ILT4 allelic frequenciesfor African, European, Asian, and South Asian populations. Sequences forhaplotypes that were expressed with a 5% or greater prevalence in anypopulation (haplotypes 1, 2, 5, 7, 9 and 10 in Table 7) were determinedand expressed in 3A9 T cells. 1E1(G4) bound all haplotypes tested usingsaturating doses of the antibody (FIG. 13). Haplotype 2 corresponded tothe consensus sequence reported in Uniprot. Haplotype 5 was used infunctional and ligand-based assays. Haplotype binding data indicatesthat 1E1(G4) binds all major allelic variants tested.

TABLE 7 Haplotype frequencies for ILT4 across human populations.rs373032 rs386056 rs7247538 rs7247451 rs1128646 p.Glu61Asp p.Val235Metp.His300Tyr p.Cys306Trp p.Arg322His Haplotype frequencies by ethnicitySOUTH Haplotype c.483A > T c.703G > A c.898C > T c.918C > G c.965G > AAll AFRICAN EUROPEAN ASIAN ASIAN 1 ATGGC D M H C R 0.23 0.09 0.15 0.600.15 2 TCGGC E V H C R 0.14 0.04 0.22 0.08 0.28 3 ATAGC D M Y C R 0.03 4TCGCC E M H W R 0.02 5 ACACT D V Y W H 0.30 0.17 0.58 0.08 0.37 6 ACACCD V Y W R 0.02 0.04 0.02 7 ACGCT D V H W H 0.02 0.06 8 ACGGT D V H C H0.02 9 ACGCC D V H W R 0.02 0.01 0.11 10 ACGGC D V H C R 0.23 0.54 0.200.06 f(SNP) 0.80 0.24 0.45 0.46 0.46*Haplotypes for each population were based on the phase 3 data from the1000 genome project and were determined using PLINK to analyzenon-synonymous SNPs listed in the EXAC database.

Example 6: RNA Expression of ILT4 in Tumor and Cell Types Based on TCGAand Blueprint Databases

Expression of ILT4 across tumor types and cell populations wasdetermined using publicly available RNAseq datasets, through Omicsoft(Qiagen, Cary, N.C.). The TOGA dataset (TCGA_B38_20171002_v4,https://gdc-portal.nci.nih.gov/) is comprised of 11,292 samples withRNA-Seq data. The Blueprint dataset (Blueprint_B38_20170216_v2,http://www.blueprint-epigenome.eu/) is comprised of 258 normal bloodsamples from 55 cell types with RNA-Seq data. The tumor types withhighest expression of ILT4, at the RNA level, include LAML (i.e., AML),DLBC (i.e., DLBCL), TGCT, MESO, KIRC (FIG. 14A). The cell types withhighest expression of ILT4, at the RNA level, include neutrophils,monocytes, osteoclasts, eosinophils, macrophages, and dendritic cells(FIG. 14B). Lymphocytes had low to no expression of ILT4, in thisdataset. FPKM of 1 (or LOG2(FPKM+0.1) of 0) is the most widely acceptedheuristic fixed threshold, although lower FPKM values could report on“low expressed” genes within a sample.

Example 7: Binding of 1E1(G4) to Myeloid Cells from Tumor HistocultureSamples

Histocultures were prepared from fresh human tumor samples (surgicalresections), and were treated with either anti-RSV IgG4 (isotypecontrol) or 1E1(G4) at 20 pg/mL for 18-24 hours at 37° C. Aftertreatment, tumor slices were digested into single cell suspensions andstained for FACS. Dot plots and contour plots represent FACS data fromeither RCC (FIG. 15A) or CRC (FIG. 15B) tumor histoculture single cellsuspensions. Total myeloid cells can be subdivided into four subsetsbased on the expression of CD66b and/or CD14. These four myeloid subsetswere simultaneously analyzed for ILT4 expression, using a non-competingcommercial anti-ILT4-PE (BioLegend, cat #338706, San Diego, Calif.), andcell surface-bound 1E1(G4), using an anti-IgG4 secondary antibody. Goodcorrelation between ILT4+ cells and 1E1(G4)+ cells was observed in1E1(G4)-treated histocultures. Tumor-infiltrating lymphocytes wereobserved to be ILT4- and 1E1(G4)- in these samples.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, the scope of the present inventionincludes embodiments specifically set forth herein and other embodimentsnot specifically set forth herein; the embodiments specifically setforth herein are not necessarily intended to be exhaustive. Variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription. Such modifications are intended to fall within the scope ofthe claims.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

SEQUENCE LISTING

The present specification is being filed with a computer readable form(CRF) copy of the Sequence Listing. The CRF entitled24443USDIV1_SEQLIST.txt, which was created on Jun. 2, 2021 126 KB bytesin size, is incorporated herein by reference in its entirety.

We claim:
 1. An antibody or antigen-binding fragment thereof that bindsone or more amino acid residues in a human immunoglobulin-liketranscript 4 (ILT4) epitope selected from the group consisting ofLYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID NO:61),and HTGRYGCQ (SEQ ID NO:62) and protects the epitope from deuteriumexchange in the presence of a deuterium source.
 2. The antibody orantigen-binding fragment thereof of claim 1, wherein the ILT4 epitope isLYREKKSASW (SEQ ID NO:59).
 3. The antibody or antigen-binding fragmentthereof of claim 1, wherein the ILT4 epitope is TRIRPEL (SEQ ID NO:60).4. The antibody or antigen-binding fragment thereof of claim 1, whereinthe ILT4 epitope is NGQF (SEQ ID NO:61).
 5. The antibody orantigen-binding fragment thereof of claim 1, wherein the ILT4 epitope isHTGRYGCQ (SEQ ID NO:62).
 6. An antibody or antigen-binding fragmentthereof that competes with a reference antibody for binding to a humanILT4 epitope selected from the group consisting of LYREKKSASW (SEQ IDNO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID NO:61), and HTGRYGCQ (SEQID NO:62), wherein the reference antibody comprises a heavy chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:2 and a light chain immunoglobulin comprising the amino acid sequenceset forth in SEQ ID NO:7.
 7. The antibody or antigen-binding fragmentthereof of claim 6, wherein the ILT4 epitope is LYREKKSASW (SEQ IDNO:59).
 8. The antibody or antigen-binding fragment thereof of claim 6,wherein the ILT4 epitope is TRIRPEL (SEQ ID NO:60).
 9. The antibody orantigen-binding fragment thereof of claim 6, wherein the ILT4 epitope isNGQF (SEQ ID NO:61).
 10. The antibody or antigen-binding fragmentthereof of claim 6, wherein the ILT4 epitope is HTGRYGCQ (SEQ ID NO:62).11. An antibody or antigen-binding fragment thereof that binds humanILT4, comprising: (1) a heavy chain variable domain comprising:(SEQ ID NO: 16) CDR-H1: GYYWS, (SEQ ID NO: 17)CDR-H2: EINHXGSTNYNPSLKS wherein X is S or A , and (SEQ ID NO: 18)CDR-H3: LPTRWVTTRYFDL;

and, a light chain variable domain comprising: (SEQ ID NO: 19)CDR-L1: TGSSSNIGAGYDVH, (SEQ ID NO: 20)CDR-L2: GX₁X₂NRPS, wherein X₁ is N, Q, E or D and X₂ is S or A, and(SEQ ID NO: 21) CDR-L3: QSFDNSLSAYV;

(2) a heavy chain variable domain comprising: (SEQ ID NO: 22)CDR-H1: SYAIS, (SEQ ID NO: 23) CDR-H2: GIIPIFGTANYAQKFQG, and(SEQ ID NO: 24) CDR-H3: YFX₁X₂SGWYKGGAFDI, wherein X₁ is D or Sand X₂ is S or A;

and, a light chain variable domain comprising: (SEQ ID NO: 25)CDR-L1: TLRSGINVDTYRIH, (SEQ ID NO: 26) CDR-L2: YKSDSDKHQGS, and(SEQ ID NO: 27) CDR-L3: AIWYSSTWV;

(3) a heavy chain variable domain comprising: (SEQ ID NO: 28)CDR-H1: SYAMH, (SEQ ID NO: 29) CDR-H2: VISYDGSNKYYADSVKG, and(SEQ ID NO: 30) CDR-H3: VGEWIQLWSPFDY;

and, a light chain variable domain comprising: (SEQ ID NO: 31)CDR-L1: RASQGISSWLA, (SEQ ID NO: 32) CDR-L2: AASSLQS, and(SEQ ID NO: 33) CDR-L3: QQYNSYPPT;

or (4) a heavy chain variable domain comprising: (SEQ ID NO: 34)CDR-H1: ELSMH, (SEQ ID NO: 35) CDR-H2: GFDPEDGETIYAQKFQG, and(SEQ ID NO: 36) CDR-H3: AGPLYTIFGVVIIPDNWFDP;

and a light chain variable domain comprising: (SEQ ID NO: 37)CDR-L1: TGSSSNIGAGYDVH, (SEQ ID NO: 38) CDR-L2: GNSNRPS, and(SEQ ID NO: 39) CDR-L3: QSYDSSLSGSGVV.


12. The antibody or antigen-binding fragment thereof of claim 11,comprising: a heavy chain variable domain comprising: (SEQ ID NO: 16)CDR-H1: GYYWS, (SEQ ID NO: 48) CDR-H2: EINHAGSTNYNPSLKS, and(SEQ ID NO: 18) CDR-H3: LPTRWVTTRYFDL;

and, a light chain variable domain comprising: (SEQ ID NO: 19)CDR-L1: TGSSSNIGAGYDVH, (SEQ ID NO: 52) CDR-L2: GDSNRPS, and(SEQ ID NO: 21) CDR-L3: QSFDNSLSAYV.


13. The antibody or antigen-binding fragment thereof of claim 11,comprising: a light chain immunoglobulin having at least 90% amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:3,4, 5, 6, 7, 11, 13, 15, or 45, and/or a heavy chain immunoglobulinhaving at least 90% amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO:1, 2, 8, 9, 10, 12, 14, 44, 79, 80, 81,82, 83, 84, 85, or
 86. 14. The antibody or antigen-binding fragmentthereof of claim 11, comprising: a light chain immunoglobulin comprisinga light chain variable domain having at least 90% amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:70, 71, 72,73, 58, 74, 75, 76, or 77, and/or a heavy chain immunoglobulincomprising a heavy chain variable domain having at least 90% amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:63,57, 64, 65, 66, 67, 68, or
 69. 15. The antibody or antigen-bindingfragment thereof of claim 11, comprising: a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:3, 4, 5, 6, 7,11, 13, 15, or 45; and/or a heavy chain immunoglobulin comprising theamino acid sequence set forth in SEQ ID NO: 1, 2, 8, 9, 10, 12, 14, 44,79, 80, 81, 82, 83, 84, 85, or
 86. 16. The antibody or antigen-bindingfragment thereof of claim 11, comprising: a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:70, 71, 72,73, 58, 74, 75, 76, or 77, and/or a heavy chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:63, 57, 64,65, 66, 67, 68, or
 69. 17. The antibody or antigen-binding fragmentthereof of claim 11, comprising: (1) a heavy chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO: 1 or 79; anda light chain immunoglobulin comprising the amino acid sequence setforth in SEQ ID NO: 3; (2) a heavy chain immunoglobulin comprising theamino acid sequence set forth in SEQ ID NO: 2 or 80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 4; (3) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 2 or 80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 5; (4) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 2 or 80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 6; (5) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 2 or 80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 7; (6) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 2 or 80; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 3; (7) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 8 or 82; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 11; (8) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 9 or 83; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 11; (9) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 10 or 84; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 11; (10) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 12 or 85; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO: 13; or (11) a heavy chain immunoglobulin comprising the amino acidsequence set forth in SEQ ID NO: 14 or 86; and a light chainimmunoglobulin comprising the amino acid sequence set forth in SEQ IDNO:
 15. 18. The antibody or antigen-binding fragment thereof of claim11, comprising: (1) a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:63; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:70; (2)a heavy chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:57; and a light chain variable domain comprising theamino acid sequence set forth in SEQ ID NO:71; (3) a heavy chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO:57; and a light chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:72, (4) a heavy chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:57; and alight chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:73; (5) a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:57; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:58; (6)a heavy chain variable domain comprising the amino acid sequence setforth in SEQ ID NO:57; and a light chain variable domain comprising theamino acid sequence set forth in SEQ ID NO:70; (7) a heavy chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO:64; and a light chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:74; (8) a heavy chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:65; and alight chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:74; (9) a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:66; and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:74;(10) a heavy chain variable domain comprising the amino acid sequenceset forth in SEQ ID NO:67; and a light chain variable domain comprisingthe amino acid sequence set forth in SEQ ID NO:75; or (11) a heavy chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO:68; and a light chain variable domain comprising the amino acidsequence set forth in SEQ ID NO:76.
 19. The antibody or antigen-bindingfragment thereof of claim 11, comprising: a heavy chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:57; and alight chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:58.
 20. The antibody or antigen-binding fragment thereof ofclaim 11, comprising: a heavy chain immunoglobulin comprising the aminoacid sequence set forth in SEQ ID NO:2; and a light chain immunoglobulincomprising the amino acid sequence set forth in SEQ ID NO:7.
 21. Theantibody or antigen-binding fragment thereof of claim 11, comprising: aheavy chain immunoglobulin comprising the amino acid sequence set forthin SEQ ID NO:80; and a light chain immunoglobulin comprising the aminoacid sequence set forth in SEQ ID NO:7.
 22. The antibody orantigen-binding fragment thereof of any one of claims 1-21, wherein theantibody or antigen-binding fragment thereof is glycosylated withengineered yeast N-linked glycans or CHO N-linked glycans.
 23. Apharmaceutical composition comprising the antibody or antigen-bindingfragment thereof of any of claims 1-22.
 24. The pharmaceuticalcomposition of claim 23, further comprises a therapeutic agent.
 25. Thepharmaceutical composition of claim 23 or 24, further comprises apharmaceutically acceptable carrier.
 26. A polynucleotide comprisingnucleotide sequence encoding a VH, a VL, both a VH and a VL, a heavychain immunoglobulin, a light chain immunoglobulin, or both a heavychain and a light chain immunoglobulin of the antibody orantigen-binding fragment according to any one of the claims 1-21.
 27. Avector comprising the polynucleotide of claim
 26. 28. A host cellcomprising the polynucleotide of claim 26 or the vector of claim
 27. 29.A method for blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/orHLA-F in a human subject in need thereof comprising administering to thehuman subject an effective amount of the antibody or antigen-bindingfragment thereof of any one of claims 1-22.
 30. A method of treating acancer in a human subject in need thereof, comprising administering tothe human subject an effective amount of the antibody or antigen-bindingfragment thereof of any one of claims 1-22.
 31. The method of claim 29or 30, further comprises performing a therapeutic procedure and/oradministering a therapeutic agent to the human subject.
 32. A method ofproducing the antibody or antigen-binding fragment thereof of any one ofclaims 1-22, comprising culturing the host cell of claim 28 to expressthe antibody or antigen-binding fragment thereof.
 33. A method ofproducing the antibody or antigen-binding fragment thereof of any one ofclaims 1-22, comprising expressing the polynucleotide of claim
 26. 34.An antibody or antigen-binding fragment thereof that binds to human ILT4which is a product of the method of claim 32 or
 33. 35. A method fordetecting an ILT4 peptide or fragment thereof in a sample, comprisingcontacting the sample with the antibody or antigen-binding fragmentthereof of any one of claims 1-22 and detecting the presence of acomplex between the antibody or antigen-binding fragment thereof and theILT4 peptide or fragment thereof; wherein detection of the complexindicates the presence of the ILT4 peptide or fragment thereof.
 36. Useof the antibody or antigen-binding fragment thereof of any one of claims1-22 for the treatment of cancer.
 37. The antibody or antigen-bindingfragment thereof of any one of claims 1-22 for use in treating cancer.38. An antibody that consists of two heavy chains and two light chains,wherein each light chain comprises the amino acid sequence set forth inSEQ ID NO:58 and each heavy chain comprises the amino acid sequence setforth in SEQ ID NO:57.
 39. The antibody of claim 38, wherein the lightchain further comprises the amino acid sequence set forth in SEQ IDNO:90 and/or the heavy chain further comprises the amino acid sequenceset forth in SEQ ID NO:89.
 40. An antibody that consists of two heavychains and two light chains, wherein each light chain comprises theamino acid sequence set forth in SEQ ID NO:7 and each heavy chaincomprises the amino acid sequence set forth in SEQ ID NO:2.
 41. Anantibody that consists of two heavy chains and two light chains, whereineach light chain consists of the amino acid sequence set forth in SEQ IDNO:7 and each heavy chain consists of the amino acid sequence set forthin SEQ ID NO:2.
 42. A pharmaceutical composition comprising: (i) anantibody that consists of two heavy chains and two light chains, whereineach light chain consists of the amino acid sequence set forth in SEQ IDNO:7 and each heavy chain consists of the amino acid sequence set forthin SEQ ID NO:2, and (ii) pembrolizumab.